Transgenic Plants: Recent Developments : Recent Developments [1 ed.] 9781622572663, 9781622572458

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Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. Transgenic Plants: Recent Developments : Recent Developments, Nova Science Publishers, Incorporated, 2012. ProQuest

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. Transgenic Plants: Recent Developments : Recent Developments, Nova Science Publishers, Incorporated, 2012. ProQuest

BOTANICAL RESEARCH AND PRACTICES

TRANSGENIC PLANTS

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

RECENT DEVELOPMENTS

No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in Transgenic Plants: Recent Developments : Recent Developments, Nova Science Publishers, Incorporated, 2012. ProQuest rendering legal, medical or any other professional services.

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Transgenic Plants: Recent Developments : Recent Developments, Nova Science Publishers, Incorporated, 2012. ProQuest

BOTANICAL RESEARCH AND PRACTICES

TRANSGENIC PLANTS

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

RECENT DEVELOPMENTS

SHEN YAO ZHU AND

JIANG LO HU EDITORS

New York

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Copyright © 2012 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com

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NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book.

Library of Congress Cataloging-in-Publication Data Transgenic plants : recent developments / editors: Shen Yao Zhu and Jiang Lo Hu. p. cm. Includes index. ISBN:  (eBook) 1. Transgenic plants. 2. Transgenic plants--Risk assessment. 3. Plant genetic engineering. I. Zhu, Shen Yao. II. Hu, Jiang Lo. SB123.57.T735 2012 632'.8--dc23 2012021146

Published by Nova Science Publishers, Inc. † New York

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CONTENTS

Preface Chapter 1

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Chapter 2

vii Plant Leaf Decomposition, DNA Release, Persistence and Transfer into the Environment John Poté and Walter Wildi Establishment of Light Formula and Light Environmental Management Strategy for High-Efficient Plant Cultivation with Artificial Light Sources Wenke Liu, Qichang Yang, Lingling Wei and Ruifeng Cheng

Chapter 3

Harvest-Inducible Genes and Promoters in Alfalfa Jian Zhang and Larry R. Erickson

Chapter 4

Toxic Impacts of Three Veterinary Antibiotics on Seed Germination and Growth as Well as Nutritional Quality of Vegetables Lian Feng Du and·Wen Ke Liu

Chapter 5

Growth Responses of Bt and Non-Bt Cottons to Soil Phosphorus, Copper and Cadmium Levels Lian Feng Du and Wenke Liu

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21

35

49

61

vi Chapter 6

Chapter 7

Contents The Application of Site-Specific Recombination Systems for Biosafety and Genome Manipulation in the Production of Transgenic Plants Yuan-Yeu Yau and Ludmila Tyler Amplification of Small Interfering RNAs in Transgenic Plants Hiroaki Kodama, Hazuki Iwasa, Sayaka Hirai and Shin-Ichiro Oka

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Index

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109

133

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PREFACE Plants whose DNA is modified using genetic engineering techniques are known as transgenic plants. In most cases the aim is to introduce a new trait to the plant which does not occur naturally in this species. Examples include resistance to certain pests, diseases or environmental conditions, or the production of a certain nutrient or pharmaceutical agent. This new book gathers and presents current research on transgenic plants including an examination of the release, persistence and transport of transgenic plant DNA in saturated and unsaturated mediums, such as soils and sediments; highefficient plant cultivation with artificial light sources; the impact of three antibiotics on seed germination, hydroponic growth and nutritional qualities of vegetables; growth responses of Bt and non-Bt cottons to soil phosphorous, copper and cadmium levels; site-specific recombination systems for genome manipulation in transgenic plants; and amplification of small interfering RNAs in transgenic plants. Chapter 1 - Purified bacterial cells and DNA, the signature of life, have been extensively studied in a wide range of environments and in different microbial ecosystems. On the contrary the release, persistence and transport of transgenic genes and the fate of transgenic DNA in different environmental compartments, have not been investigated in sufficient detail and depth, and are less well documented. Modelling DNA release from plant material may help to understand to the destiny of released plant DNA (transgenic or non transgenic) and its dispersion into the environment. The question of the environmental impact and risk of transgene transfer from genetically modified plants (GMPs) to the natural environment remains a subject of intense scientific and also political debate. Thus, the questions concerning the fate of transgenes in the environment are not easy to answer,

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Shen Yao Zhu and Jiang Lo Hu

since the mechanisms and parameters controlling these processes are complex and not completely understood. The deliberate or accidental release of genetically engineered microorganisms or antibiotic resistance genes from transgenic plants into the environment may be a possible source of biological contamination of soil, surface and ground water. The main potential risk of the presence of recombinant DNA (rDNA) in the environment is the persistence of transgenes released from transgenic plants and their transport into different environmental compartments. Consequently, the ability of the transgenes to transform competent soil or aquatic microorganisms is a key question. This chapter is part of a wider study examining the release, persistence and transport of transgenic plant DNA in saturated and unsaturated mediums, such as soils and sediments. The kinetics of plant leaf mass loss and DNA release as well as the long-term physical persistence and biological activity of transgenic genes such as aadA and blaTEM marker genes from transgenic plant DNA are developed. The data presented here suggest that plant leaf decomposition and DNA release may be described by a two-compartment first order model. This model indicates a potential for biologically active DNA to be transported over considerable distances in saturated and unsaturated medium. Additionally, the data demonstrate the persistence of DNA in the environment over long time periods and provide new insights into the fate of transgenic plant DNA in soil and aquatic environments. They also put an accent on the question regarding the possible ecological implications of the presence of extracellular transgenic DNA in groundwater. Chapter 2 - Light is a vital environmental factor that affects plant growth and development by acting on plants not only as the energy source for photosynthesis, but also as an environmental signal for photomorphogenesis. Light requirements of plants are subjected to species, cultivar, growth and developmental stages, environmental conditions and manipulation target of yield and quality. Therefore, detailed studies on light quality requirements, i.e. light fomula (LF), based on biological and physiological requirement are urgently needed for getting high yield and good quality of protected plants. With the development of semiconductor solid light sources, light-emitting diodes (LEDs), detailed research on plant biology and physiology responses tomonochromatic light became possible. Therefore, the light quality requirements for all kinds of plants can be precisely and extensively studied to obtain the optimal light spectrum component for high productivity and good quality. The LF was defined as optimized monochromatic light component illuminated by LEDs for getting higher biomass and better nutritional quality of plants. Based on published literatures, red and blue light spectrum are

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ix

macro-necessary LF components, and purple, green, yellow, cyan, orange and other visible light spectra are micro-beneficial LF components, while far-red and ultraviolet light spectrum is functional LF components. Generally, a kind of LF is composed of one or two kinds of necessary light spectra, several micro-beneficial and functional light spectra for special plant during a certain period and under certain environmental conditions. The LF is of crucial scientific significance that should be established for plants grown with artificial light. More importantly, the LF is an important part of light environment management strategy (LEMS). The LEMS refers to a comprehensive management method of light environment, including light intensity, LF and photoperiod for one special plant, which should be established for plants with artificial light sources. It is assumed that the LF and LEMS will be preferentially applied in plant factory with entire artificial light source. Chapter 3 - The harvesting and storing of alfalfa is a routine practice in the agricultural industry worldwide. A study of the physiological and biochemical changes that occur after harvesting may help to understand how plants respond to this process. To investigate gene expression in harvested alfalfa (Medicago sativa), cDNA from non-harvested and harvested plants in the field was subjected to subtractive hybridization to identify, in particular, those genes that are induced by the harvesting treatment. Three different genes, named, hi7, hi11 and hi12, were isolated and analysed. Northern blot analysis confirmed that hi7, hi11 and hi12 are strongly induced during a post harvest incubation period. The promoters of these genes were isolated and characterized. Reporter gene driven by the promoter has been transformed into transgenic plants. The hi12 promoter showed it is harvesting inducible. Chapter 4 - Two experiments were conducted to investigate the impacts of three antibiotics on seed germination, hydroponic growth and nutritional qualities of vegetables. Seed germination test of three vegetables (cucumber, rape and Chinese cabbage) with six concentrations of tetracycline (TC), chlorotetracycline (CTC) and oxytetracycline (OTC) in form of hydrochlorides was carried out to study the impacts of these antibiotics on root elongation. Hydroponic experiment was designed to explore the growth inhibition and nutritional quality responses of lettuce grown in greenhouse with presence of various concentrations of CTC and OTC. The results showed that three antibiotics inhibited root elongation of germinated seeds, and the inhibitory rates depended on the drug species, antibiotic concentrations and vegetable species. Shoot and root biomass of lettuce were significantly reduced when

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Shen Yao Zhu and Jiang Lo Hu

antibiotics added in the nutrient solution. In addition, the soluble sugar content in leaves and petiole, and soluble protein in petiole were modified by antibiotic pollution. To conclude, antibiotic pollution of soil and water will adversely impact the seed germination and growth of protected vegetables grown in soil or hydroponics. Chapter 5 - Two glasshouse experiments were conducted to investigate the growth differences of Bt- and non-Bt cotton on soils applied with various phosphorus (P), copper (Cu) and cadmium (Cd) levels. The results showed that high P level had no significant effect on shoot dry weight, root length and root dry weight of non-Bt cotton compared with low P treatment. However, high P level increased the shoot dry weight, root length, root dry weight and specific root length of Bt cotton. There was no difference between biological indices except specific root length between Bt cotton and non-Bt cotton under high P condition. Soil Cd and Cu addition inhibited the growth of both Bt and non-Bt cotton, with a significant decrease in shoot height, shoot dry weight and root dry weight. However, non-Bt cotton presented higher shoot height and shoot dry weight at the higher Cd and Cu levels. Chapter 6 - By enabling the development of new crop varieties with increased nutritional content, disease resistance, and stress tolerance, plant biotechnology plays an important role in addressing the food needs of the world’s population. According to ISAAA Brief 39-2008, the area of land used globally for cultivating transgenic crops grew from 114.3 million hectares in 2007 to 125 million hectares in 2008. In 2007, the direct global farm income benefit from biotech crops was $10.1 billion. So far, the majority of transgenic crops have been produced either by Agrobacterium-mediated transformation or by biolistic transformation. The gene controlling a trait of interest is usually delivered along with a selectable marker gene (such as an antibiotic-resistance gene) for later selection of the transgenic plants from the bulk of nontransformed plants. After serving this purpose, the selectable marker gene is no longer needed. However, this approach for producing transgenic crops has raised concerns from both the regulatory and public sectors over the presence of selectable marker genes in the food supply or their possible escape into the environment through the outcrossing of transgenic crops to related species. Therefore, research in plant transgenesis continues to focus on ways to generate transgenic crops free of selectable marker genes. Several approaches for producing selectable-marker-free transgenic crops have been reported. Among these approaches, one of the most powerful is the application of microbial site-specific recombination systems to the transformation technology. This article will discuss the promise of transgenic breeding for

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future agriculture, the public concerns over genetically modified plants, and the utilization of site-specific recombination systems (including the novel ParA system recently described by our laboratory) for the production of marker-free transgenic plants. The use of site-specific recombination systems to tailor controlled transgene expression in plants and to genetically engineer plants for breeding programs will be discussed, as well. Chapter 7 - RNA interference (RNAi) is a rapidly popularized technology which contributes to progress in basic plant sciences and applied plant biotechnology. Most plant RNAi vectors transcribe an inverted repeat sequence consisting of target sequences. The resultant hairpin RNAs are processed into 21~25-nucleotide-long small interfering RNAs (siRNAs). These primary siRNAs are incorporated into the ARGONAUTE1-containing protein complexes, and then guide the sequence-specific cleavage of mRNAs. Primary siRNAs often trigger the synthesis of secondary siRNAs. In this amplification process, annealing of primary siRNAs to target mRNAs is followed by recruitment of RNA-DEPENDENT RNA POLYMERASE6 (RDR6), and then complementary RNA molecules against the target mRNAs are synthesized. The newly synthesized dsRNAs are processed into secondary siRNAs by redundant activities of DICER-LIKE4 and DICER-LIKE2. Since the target region of secondary siRNAs spreads into regions outside the primary target site, this phenomenon is called transitivity. Spreading of RNAi target via transitivity deteriorates specificity of RNAi. Therefore, elucidation of mechanistic issues of transitivity is important for researchers who use RNAi plants. Transitivity is frequently observed in transgenic plants expressing target transgene sequences under the presence of primary siRNAs, and there is only one report showing transitivity along the endogenous mRNA templates. It remains unclear why most endogenous transcripts seem to be an inefficient template for RDR6 during transitivity, but the limited transitivity along the endogenous transcripts apparently contributes to the maintenance of specificity of RNAi. This review is focused on transitivity observed in transgenic plants.

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In: Transgenic Plants Editors: Shen Y. Zhu and Jiang L.Hu

ISBN 978-1-62257-245-8 ©2012 Nova Science Publishers, Inc.

Chapter 1

PLANT LEAF DECOMPOSITION, DNA RELEASE, PERSISTENCE AND TRANSFER INTO THE ENVIRONMENT John Poté and Walter Wildi

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University of Geneva, Institute F.A. Forel and Institute of Environmental Sciences, Versoix, Switzerland

ABSTRACT Purified bacterial cells and DNA, the signature of life, have been extensively studied in a wide range of environments and in different microbial ecosystems. On the contrary the release, persistence and transport of transgenic genes and the fate of transgenic DNA in different environmental compartments, have not been investigated in sufficient detail and depth, and are less well documented. Modelling DNA release from plant material may help to understand to the destiny of released plant DNA (transgenic or non transgenic) and its dispersion into the environment. The question of the environmental impact and risk of transgene transfer from genetically modified plants (GMPs) to the natural environment remains a subject of intense scientific and also political debate. Thus, the questions concerning the fate of transgenes in the environment are not easy to answer, since the mechanisms and 

Corresponding author. Tel.: +41-22-379-03-21; Fax: + 41 22 379 03 29. E-mail: [email protected].

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John Poté and Walter Wildi parameters controlling these processes are complex and not completely understood. The deliberate or accidental release of genetically engineered microorganisms or antibiotic resistance genes from transgenic plants into the environment may be a possible source of biological contamination of soil, surface and ground water. The main potential risk of the presence of recombinant DNA (rDNA) in the environment is the persistence of transgenes released from transgenic plants and their transport into different environmental compartments. Consequently, the ability of the transgenes to transform competent soil or aquatic microorganisms is a key question. This chapter is part of a wider study examining the release, persistence and transport of transgenic plant DNA in saturated and unsaturated mediums, such as soils and sediments. The kinetics of plant leaf mass loss and DNA release as well as the long-term physical persistence and biological activity of transgenic genes such as aadA and blaTEM marker genes from transgenic plant DNA are developed. The data presented here suggest that plant leaf decomposition and DNA release may be described by a two-compartment first order model. This model indicates a potential for biologically active DNA to be transported over considerable distances in saturated and unsaturated medium. Additionally, the data demonstrate the persistence of DNA in the environment over long time periods and provide new insights into the fate of transgenic plant DNA in soil and aquatic environments. They also put an accent on the question regarding the possible ecological implications of the presence of extracellular transgenic DNA in groundwater.

Keywords: Plant DNA; transgenic plants; DNA release; Antibiotic gene resistant; persistence; degradation; transport, exponential model

1. INTRODUCTION The global agricultural surface cultivated by genetically modified plants (GMPs) continues to increase. In 2010, more than 120 million hectares of GMPs were sown world wide. Herbicide tolerance and microbial insect resistance properties are present in all of the major GMPs including maize, soybeans, rapeseed and cotton. These GMPs are grown mainly in the United States, Argentina, Canada and Brazil. The herbicide-tolerant soybeans account for about 78.6% of the world’s biotech crops [1]. Technological innovations, such as GMPs, are considered bringing their own set of benefits and potential risks for the environment. Therefore, the new transgenic species incorporated in crops should be evaluated at least for environmental safety, food security

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and ethical aspects [2]. One of the main risks of transgenic plants is the release and dispersal of their transgenes, before they are degraded, through gene flow into the environment and into wild populations. Ecological effects may be caused by deliberate or accidental release of genetically engineered microorganisms, first by survival, then by multiplication and dispersal of the inoculated cells as well as their transfer of recombinant DNA (rDNA) to indigenous microorganisms; these organisms have therefore to be monitored in the environment [3]. Additionally, the transgenic plants may spread their transgenes through gene flow into wild populations causing genetic pollution and giving rise to potential ecosystem disruption. The following issues associated with genes from transgenic plants must be dealt with: (i) emission, dispersal, and deposition of transgenic pollen, (ii) introgression of the transgene into wild species, (iii) stabilisation and spread of the transgene in wild species, (iv) ecological effects of the transgene in the new host population [4]. DNA from plants has been released for very long times into the environment naturally [5, 6]. According to different studies, the principal ways of transgene release from GMPs and their entry into the environment were summarised [7,8]: (i) by air transport (mainly through pollen dispersal), (ii) by decay in the residuesphere, i.e. enzymatic degradation of cell structures by plant pathogens and release of DNA, followed by infiltration into the soil and possibly into the groundwater, or runoff towards the surface water, (iii) by degradation of plant debris incorporated in the soil, followed by infiltration, or runoff towards the surface water, (iv) by degradation and infiltration from the rhizosphere (sloughing of root cap cells) into the soil, surface water and possibly to the groundwater. Cell lyses during plant material decomposition can be considered as the main mechanism participating in the release of plant DNA to the environment [3, 9-12]. Studies regarding the assessment of the potential risk of the use of transgenic plants in agriculture are mostly focussed in the fate of released recombinant DNA into the environment. However, according to many scientific publications [8, 12, 13-20], rDNA released during plant material decomposition in the soil, sediments and surface water is generally poorly characterized. Also, little is known about the movement, temporal or spatial dispersal of rDNA in subsurface soil and fate of rDNA in the aquatic environment. It is thus important to focus further research efforts on these aspects. Some studies performed on plant material decomposition demonstrated that (i) plant material decomposition in the soil is temporally limiting the transfer of DNA to the soil; before release, important quantities of DNA is

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degraded inside the plant tissues during decomposition [17], (ii) plant DNA release, long term persistence and the movement of DNA towards the vadose zone of the soil and to the groundwater [18, 19]. On the other hand, field data demonstrated that many of the genes such as blaTEM116 gene (encoding resistance to ampicillin, which belongs to the beta-lactam antibiotic family), and aadA genes (encoding resistance against to spectinomycin and streptomycin) are incorporated into GMPs. These genes are present naturally in the environment, as well as soil bacteria, and can be naturally resistant to a broad spectrum of antibiotics [12, 16, 20]. However, depending on environmental factors, further attention is needed with respect to these aspects concerning long-term cropping of GMPs. The assessment of the environmental impact of rDNA from GMPs, and in particular its kinetic aspects, requires the use of models capable of assessing the characteristics of the initial source, plant material decomposition processes, rDNA release processes, the means of transport and the interaction of rDNA with biotic and abiotic parameters of the environment [11, 17, 21].

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2. FATE OF DNA INTO THE ENVIRONMENT While DNA analysis has become central to work on natural gene exchange, forensic analyses, soil bioremediation, genetically modified organisms, exobiology, and palaeontology, fundamental questions about DNA resistance to degradation remain. The Earth is virtually entirely covered in nucleic acid as well as DNA from plants has been released into the environment naturally [6]. Thus, nucleic acids are ubiquitous in many environments, including fresh water, marine and surface water column and sediments, soil and terrestrial subsurface [9, 22, 23]. The fate of DNA in the environment can be summarized as follows: (i) DNA release from organisms (plant, microorganisms, animals), (ii) persistence of extracellular DNA according to environmental parameters; (iii) adsorption of extracellular DNA to the soil matrix; (iv) degradation of extracellular DNA by DNases; (v) extracellular DNA transforms competent soil microorganisms (binding of DNA onto soil components does not eliminate the ability of bound DNA to transform competent soil microorganisms [6, 12, 13]); (vi) probable dispersal and vertical movement of extracellular DNA in unsaturated soil medium; (vii) extracellular DNA used as nutrient source by soil and aquatic microorganisms. Although, data about the frequency or likelihood of each of these steps is available, the entire sequence is difficult to monitor in the field. During these

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last years, many studies have been performed in field and microcosms to describe these processes [8, 24-26]. After released from organisms in the environment compartments described above, eDNA can be rapidly adsorbed to soil or sediment particles. Numerous experiments have demonstrated the mechanisms and importance of DNA adsorption onto soil components such as sand particles, clays minerals, or humic compounds [9, 28-32]. These results suggest that free DNA is rapidly degraded in the environment, but the adsorption of DNA onto soil components retards DNA degradation and constitutes a major mechanism of DNA molecule persistence in soil. However, a noticeable aspect of the majority of these studies is that the quality and quantity of DNA in plant materials (during decomposition) before release into different environments should be investigated.

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2.1. Fate of Extracellular DNA in Aquatic Environment The type of aquatic environment affects the amount of DNA present and what can be recovered using various extraction methods [32]. The surface and marine water concentration of dissolved eDNA has been estimated to range from 2 to 90 g L-1 [27-31]. In the sediments from Lake Geneva, the eDNA concentration ranged from 15.3 to 22.4 μg g-1 [21]. Extracellular DNA can be used as C, N and P sources by heterotrophic microorganisms and plays a significant role in bacterial biofilm formation in the sediments. It is therefore considered as a source of nutrient and genetic information in aquatic environment [26]. It has been demonstrated that eDNA can be adsorbed onto sediment components, escapes from enzymatic degradation or chemical destruction and play a part important in horizontal gene transfer (HGT) [30, 32, 33-35]. The fate of eDNA in groundwater aquifer is poorly investigated. The concentration of eDNA in Geneva groundwater range from 5.3 to 7.8 g L-1 [19]. Numerous experiments have demonstrated the mechanisms and importance of DNA adsorption onto sediment components such as sand particles, clays minerals, or humic compounds. These results suggest that free DNA is rapidly degraded in the environment, but the adsorption of DNA onto sediments components retards DNA degradation and constitutes a major mechanism of DNA molecule persistence in the sediments. However, noticeable aspects of the majority of these studies are that the quality and quantity of rDNA in plant materials (during decomposition) before release, the

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potentiality of rDNA (after released in both sediments and water column) to transform competent bacteria in the aquatic compartments are lacking.

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2.2. Persistence of Transgenic DNA in Aquatic Environment The processes resulting from transgenic plants in agriculture fields such; pollen dispersal, decomposition of plant material in soil and DNA release, water flow and rDNA transport in vadose zone, presence of transgenic plant crops near surface waters, the input of crop byproducts in the streams, had promoted studies on the fate of transgenic DNA in the aquatic environment. These studies are summarised in the many literatures [13, 14, 19, 21, 26, 33]. It has been demonstrated that DNA from transgenic plant can persist in aquatic environment and increases the probability of horizontal gene transfer in microorganisms by transduction, transformation and conjugation. But according to the water characteristics, important quantities of DNA are degraded rapidly in water column. Bacteria transformation can only occur if the bacteria cell is in a particular physiological state named competence. The development of competence is usually monitored by detecting bacterial transformation, which involves binding of exogenous DNA on the external surface of the bacterial cell, DNA uptake usually as a single strand, insertion of the single strand in the chromosome or double strand plasmid reconstruction and gene expression [26]. Thus, the studies performed using extracellular purified rDNA (plasmid and chromosomal transgenic DNA) in microcosms to monitor its persistence and transformability in water concluded that no antibiotic or bacteria transformation were occurred under their experimentation conditions [32-35]. Some studies have been performed in field to monitor the persistence of rDNA from transgenic Bt corn (cry1 Ab gene) into aquatic environment [13, 14]. Their data suggest the persistence of transgenes in water and sediments as well as its potential impacts to aquatic leaving organisms. These researches confirmed that transgene expression and bacteria antibiotic resistance and transformability in water column and sediments are not sufficiently investigated to confirm many of the hypotheses. Therefore, they propose that further research and monitoring efforts on the identity of rDNA in aquatic environment are required.

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3. PLANT MATERIAL DECOMPOSITION AND DNA RELEASE

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Plant materials are one of the principal source inputs of organic nutrients such as carbon, nitrogen, and phosphorus in aquatic environment. Prediction of plant material decomposition in the environment is correlated to the release of macromolecule content including cellulose, holocellulose, lignin, tannins and DNA [17, 33, 34]. The decomposition of plant material in an ecological system can be determined using a variety of methods including mass loss, variation in chemical composition, changes in C/N ratio, CO2 emission, and release of molecules and elements [38-40]. The model commonly used to describe the decomposition of plant litter in soil is composed of the single (Eq. 1) and double exponential (Eq. 2) decay functions [41-43]: MDRY(t) = MDRY0e-kDRY·t

(1)

MDRY(t) = Ae-ka·t + Be-kb·t

(2)

where MDRY(t) is the dry matter weight at time t, MDRY0 is the initial dry matter weight, and kDRY is the loss rate constant (decomposition rate), A and B are the initial dry matter proportions of the fast and the slow decomposing compartments, respectively (MDRY0 = A + B), and ka and kb are respectively the decay rate constants for A and B fractions. Each fraction decreases exponentially. The half-decomposition time of the dry matter is given by:

TDRY 1 / 2 

1 kDRY

ln 2 (3)

Several factors such as structural composition of leaves, microbial activity, invertebrates, as well as temperature and humidity play a key role in the decomposition of plant materials in the environment. It has been demonstrated that the DNA mass loss concentration is not constant in the dry matter of leaves, showing decrease through time [15, 17]. Therefore, DNA weight can be determined from the measurement of the DNA mass concentration in leaves, CDNA(t), and the previous dry matter weight of samples MDRY(t), using the direct relation:

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M DNA (t )  CDNA (t )  M DRY (t )

(4)

This equation (Eq. 4), an exponential model fitting, allows the determination of both kDNA and the associated DNA half-decrease time given by:

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TDNA 1 / 2 

1 k DNA

 ln 2 (5)

Interests in the fate of recombinant DNA in the environment, many studies have been performed in field and microcosms on the decomposition of plant material and DNA release in the soil [10, 11, 17, 44-46]. The results indicate that the decrease of dry matter in leaves in both varieties was better described by a two-compartment (double exponential) first order model. Flores et al. [47] compared the decomposition of various transgenic Bt and non-Bt plants. The authors concluded that the biomass of transgenic Bt plant was decomposed less in soil than the biomass of their near-isogenic non- Bt plant counterparts. The authors recommended others studies to clarify the environmental impacts of lower degradation of the biomass of Bt plant; RosiMarshall et al. [13] measured breakdown rates of Bt and non-Bt corn litter to determine whether the Bt δ-endo-toxin influences rates of organic matter processing in their study stream. They found that no difference in decomposition rates between Bt and non-Bt corn suggesting that transgenic and nontransgenic corn byproducts are processed in the same manner in stream ecosystems; Levy-Booth et al. [15] monitored the decomposition of roundup ready leaf biomass in the soil microcosms. The soybean biomass decomposition was described using a single-phase exponential equation (eq. 1). The authors founded that the biomass of roundup ready soybean leaves was 8.6% less than non-transgenic soybean leaves after 30 days in the soil microcosms, and concluded that their study was not an investigation of DNA entry pathways under field conditions, but a model system to investigate the entry of DNA in a controlled environment. The data concerning the decrease of transgenic DNA in decomposing plant, monitoring and quantifying the transgenes in aquatic environment remain lacking. Some published data [21] performed in field and microcosms to investigate these aspects in aquatic environment have been performed using non transgenic tomato leaves. For the perspectives, the obtained results recommended the use of transgenic plants.

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9

Further extension and application of these results should be performed with specific transgenes. The possibility of bacteria antibiotic resistance and the horizontal gene transfer to sediment microorganisms is also worthy of investigation. The relative contribution of various plant communities (e.g. sediment bacteria or endo/exophytes) in facilitating extra versus intracellular plant DNA degradation also needs to be examined. Fulfilling of data could provide on the examination of both the quantitative and qualitative aspects of rDNA in plant material decomposition in the water column and sediments. Quantitative measurement includes rDNA concentration in planta and in water and sediments after released. Qualitative measurements include both the degradation of rDNA and its potential to transform bacteria leading to the production of antibiotic resistant bacteria.

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4. WATER FLOW AND DNA TRANSPORT IN SUBSURFACE ENVIRONMENT The unsaturated zone is inextricably involved in many aspects of hydrology: infiltration, vertical advection by water capillarity, evaporation, groundwater recharge, soil moisture storage, and soil erosion [48]. It also contributes to the spatial and temporal distributions of plant communities under naturally occurring rainfed conditions and serves as a modifying influence on the production of cultivated crop species. Few studies have investigated the simultaneous processes of plant material degradation, transgenic DNA release and transport by water in unsaturated and saturated zones, and reach aquatic environment such as groundwater, water column and sediments. Poté et al. [22] used extracted and purified DNA (plasmid pLEPO1 which confers resistence to spectinomycin and streptomycin) to study the transport of antibiotic genes in saturated and unsaturated soil medium using the laboratory columns. Contact between bacteria and the exogenous DNA was improved by mixing the soil and DNA in a microcosm with the result that bacteria were able to incorporate DNA in soil. These results suggest a potential for biologically active DNA to be transported over considerable distances in saturated soil and ground water. Ceccherini et al [24] used unsaturated soil column to study the vertical advection of 35S-nptII by water capillarity. The results show the vertical movement of recombinant DNA due the capillary rise and suggest the possibility of DNA degradation within the soil column. The results of these

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laboratory studies suggest that when extracellular DNA enters the soil environment, it might be dispersed by rain, irrigation-induced percolation, runoff or groundwater before being completely degraded. Gulden et al. [25] were able to quantify the amount of corn and soybean DNA released by plant roots into leachate water during growth and early plant decomposition in water. They showed that detectable quantities of plant target DNA were released into the soil environment and were moved by water during growth and early decomposition of roots. Poté et al. [18] used non transgenic tomatoes to simulate the natural processes of DNA release and vertical movement of released DNA in vadose zone using unsaturated soil column. The results suggest that DNA could be transported in subsurface soil and can reach the groundwater. Water flow and rDNA dispersal in the environment concerns both the processes of transgenes release from GMPs and the evolution of active bacteria capable of incorporating this DNA into their genomes. Many literatures report on the widespread occurrence of plant DNA (non transgenic plant) in groundwater and traditional drinking water fountains in the Geneva Champagne Basin [19]. The observation supports the notion of plant DNA release, long term persistence and movement in the unsaturated medium as well as in groundwater aquifers.

5. ANTIBIOTIC RESISTANCE BACTERIA AND HORIZONTAL GENE TRANSFER The antibiotic resistance bacteria (ARB), the transfer frequency of transgenes from plants to environmental bacteria and expression of these genes in these new bacterial hosts are considered as crucial issues when evaluating the impact of transgenic plants. Several studies indicate that such transfers are possible, and that these transfers are directly related to the transgenic status of these plants. Plants subjected to infection by pathogens are efficiently colonized by other soil micro-organisms, including some bacteria that develop genetic competence and are transformed by exogenous DNA including this released by the transgenic plant [49, 50]. The same group showed that a decaying plant suspension (residuesphere) was another hot spot for gene transfer between transgenic plants and colonizing soil bacteria [51]. When decaying, plant releases DNA in contact to metabolically active bacteria due to the presence of nutriments that permits bacterial growth and development of a

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Plant Leaf Decomposition, DNA Release, Persistence and Transfer ... 11 genetic competence stage. However, plant-bacteria DNA transfer was detected only when the recipient bacteria contained the plant DNA regions flanking the marker gene on which homologous recombination could be initiated, indicating that the main barrier to gene transfer is not physical or physiological but genetic [49, 51-52]. For years, gene transfer between transgenic plants and bacteria was considered as theoretically possible but the lack of experimental confirmation led some scientists to conclude that such events are very unlikely. Consequently, several questions remain regarding gene transfer from plants to bacteria: (i) Can any prokaryote sequence cloned in the plant genome able to be transferred to any soil or sediment bacteria? Can these plant sequences be transferred at the same frequency or does the transfer frequency depend on DNA sequences prevalence on which homologous recombination can occur among soil and sediment bacteria? (ii) Can these prokaryote sequences be involved in the co-transfer of flanking DNA regions in the plant genome and what is the frequency of such specific events? (iii) Can natural genetic transformation the only mechanism that soil bacteria use to acquire transgene genes? (iv) Are transgenic plants-bacteria DNA transfer events limited to some environmental “hot spots” or do they also happen in the bulk soil? (v) What is the occurrence frequency of these plant-to-bacteria transfer events in these various environments? These questions require the development of specific tools to discriminate between independent transfer events and clonal multiplication of initial transformants by the researches under in vitro conditions or/and in the field. Specific technical improvements are also necessary to determine if transfer events can occur under natural conditions requiring detecting specific transgene molecular signatures in bacteria genomes recovered from soil samples collected in a field planted with transgenic plants. Another important question is related to the nucleotide similarity level that would potentially exist between plant transgene (donor DNA) and soil bacteria genomes (recipient organisms), a criterion susceptible to strongly regulate recombination mediated transgene gene integration in bacteria. For any plant transgene whose nucleotide sequence is available in public database experimental and in silico studies would be necessary to determine the actual potential of these transgenes to be fully or partly transferred to bacteria. These include soil or sediment metagenome sequence analysis to detect bacteria sequences similar to those of the transgenes and transformation tests in model bacteria to assess ability of these bacterial sequences to promote recombination with plant transgene genes. The studies should not be restricted

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to typical “agronomic” transgenes (cry, pat) but should also include those whose genes code for bio-active pharmaceutical molecules considering that their potential transfer to soil or sediment bacteria could have a stronger environmental or sanitary impact than agronomic transgenes.

6. A BRIEF PROTOCOL TO STUDY THE FATE OF RDNA IN TO THE AQUATIC ENVIRONMENT The experiments for studying the fate of transgenic DNA into the aquatic environment can be performed in the microcosms (Figure 1) and different analyses can be performed as summarized in Figure 2. The study consists in 3 phases; (i) Decrease of transgenic DNA in planta during plant material decomposing in sediments, (ii) persistence of transgenic DNA in water column and sediments, and (iii) antibiotic-resistant bacteria (ARB) and horizontal gene transfer (HGT). These microcosms present several advantages:

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 

 



No input relative pollution into the lake when using different substances the relatively large size of the equipments and mass of associated environmental media minimises the effect of small scale variation in the system by maintaining a natural ecosystem in the microcosms both biological and chemical interactions between water and sediment can be studied maintaining and stabilising the physicochemical parameters, pH, temperature, conductivity,…. of water at the fixed values throughout the experimentation mimics the environment recuperation and treatment of contaminated leachates discharged

6.1. Decrease of Transgenic DNA in Planta during Plant Material Decomposing in Sediments Plant Leaf Mass Loss According to our preliminary tests using non transgenic tobacco, the experimentation for plant material mass loss can be conducted for a total of 40 days (complete degradation of leaves).

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Figure 1. Simplified picture of platform microcosm developed at Forel Institute, University of Geneva to simulate lake conditions for studying the fate of pollutants in aquatic environment.

Figure 2. Simplified protocol for different analysis steps. The experiment can be conducted in triplicate in each set of conditions (three replicates by sampling point). The sediment can be sampled after complete degradation of leaves. Water has to be sampling in continuous-flow.

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Leaves can be taken from the microcosms (Figure 1) after e.g. 1, 2, 5, 15, 25, 30 and 40 days (each container at sampling point with three replicates). Tobacco leaves can be removed from the microcosms and the nets dried at 30 °C for 5 days, and then weighed (Figure 2). Care has to be taken to remove all pieces of leaves attached to the net [21]. The results will be expressed as the mean of the triplicates with a standard deviation, and fitting models can be performed using SigmaPlot-9. The decomposition rate (modelling plant biomass) can be determined by fitting an exponential model to dry weight measurements of biomass loss and fitting this data to the decay time equations (Eq. 1-3).

Identity of Transgene in Planta To determine the identity of transgene in planta during transgenic plant material decomposing in sediments, the quantitative and qualitative aspects of transgene have be determined in the same different sampling points as described above. Plant DNA will be extracted from the same samples of plant material sampled from sediment microcosms (Figure 1). The quantitative aspects of transgene include the concentration of total DNA and the number of transgene copies can be performed using real time PCR. Qualitative aspects include the relative degradation of transgene in agarose gel and the transformation frequencies indigenous bacteria such as for E. Coli DH 10B and Acinetobacter sp. BD 413 [12, 22, 50]. The identity of transgenes can be monitored using linear regression of the mean gene copies/g leaf biomass over time: G = e-kt+ [gene]t=0, Where G: gene copies/ g dry plant material biomass, k: degradation coefficient, t: time in days and [gene]t=0 gene copies/plant biomass at the time of incubation. The decrease of transgene in plant material decomposing in sediments can be modelling using Eq. 4 and 5.

6.2. Persistence of Transgenic DNA in Water Column and Sediments For this task, two series of microcosm experimentations can be performed. The first consists to introduce 10 to 15 g of dry transgenic leaves in water column. These microcosms will serve to monitor the identity of released rDNA in water column. The leachates will be collected in continuous-flow in sterile polyethylene bottles. The analysis can be performed after e.g. 10, 20, 40, 60, 80 and 100 days, which correspond to 5-10 L of leachate water per

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Plant Leaf Decomposition, DNA Release, Persistence and Transfer ... 15 sampling time. Before analyses (e.g. DNA extraction), water samples have to be concentrated more than 200-1000 times using per example Kühner Lyophilisateur at – 45°C and by Speed Vac plus SC 110A. These operations can not affect the quantity and the quality of DNA [19, 28]. DNA from concentrated water will be extracted and purified following the procedure described previously [18, 19, 27, 28]. For the second experimentation, the microcosms will be performed as described above §5.1. After 40 days (complete degradation of leaves), sediments can be sampled in several points of microcosms (by removing delicately the top layer of sediment 0-1 cm ) as described in our previous studies [21]. All extracted DNA will therefore serve to monitor the quantitative and qualitative aspects of transgene in water column and sediments [12, 16, 22].

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6.3. Antibiotic-resistant Bacteria and Horizontal Gene Transfer The use of antibiotic resistance genes in the development of GMPs has given rise to environmental concern. Knowledge about the prevalence and diversity of the antibiotic resistance gene sediment bacteria communities is required to evaluate the possibility and ecological consequences of the transfer of these genes from GMPs to sediment bacteria. Our recent studies demonstrated that plant transgene genes that would have been naturally transferred into soil representatives under field conditions cannot be easily detected in soil bacteria because of the low frequency of transfer occurrence in nature, of the impossibility of extracting bacteria and DNA from large samples of soil and of cultivating in vitro more than a limited proportion of these bacteria. For example, the aadA gene conferring resistance to spectinomycin and streptomycin is the transgenes commonly used in transplastomic genetically modified plant constructions and the most common betalactamases are the TEM beta-lactamases that are encoded by the blaTEM1 gene and its descendants. The investigation of bacteria resistant against these antibiotics and the possibility of transgenes from GMPs transfer to bacteria has been largely investigated in our previous studies [20, 24]. According to the studies performed in the field soil conditions and microcosms, the development of methodologies for the detection of such events is complex and remains very difficult. However, these approaches are fundamentals and essentials to the evaluation of potential gene transfer and ecological consequences of transgene from GMPs into the environment [10, 11, 20, 30].

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CONCLUSION There is a paucity of information regarding the release and identity of transgenic DNA and its biological impacts in aquatic environment. The interdisciplinary approaches, e.g. rDNA release, persistence and bacteria antibiotic resistance and HGT, and their correlation with sediment and water characteristics are very important and can be of high interest to the international scientific community. Numerous questions remain about the role that natural environments play in the maintenance and dispersion of antibiotic resistance microorganisms and about the frequency with which the genes are exchanged among indigenous bacteria in aquatic environments.

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[19] Poté, J., Mavingui, P., Navarro, I., Rosselli, W., Wildi, W., Simonet, P., Vogel, T.M., Extracellular Plant DNA in Geneva Groundwater and Traditional Artesian Drinking Water Fountains. Chemosphere, 75, 498-504. [20] Demanèche, S., Sanguin, H., Poté, J., Navarro, E., Bernillon, D., Mavingui, P., Wildi, W., Vogel, T-M., Simonet, P. 2008. Antibioticresistant soil bacteria in transgenic plant fields. PNAS. 105, 3957-3962. [21] Poté, J., Ackermann, A., Wildi, W., Plant leaf mass loss and DNA release in fresh water sediments. Ecotox. Environ. Safety, 1378-1383. [22] Poté, J., Ceccherini, M.T., Tran Van, V., Rosselli, W., Wildi, W., Simonet, P., Vogel, T.M., 2003. Fate and Transport of Antibiotic Resistance Genes in Saturated Soil Columns. Eur. J. Soil Biol. 39, 65-71. [23] Nielsen, D.R., Genucheten, M.TH., Biggar, J.W., 1986. Water flow and solute transport processes in unsaturated zone. Wat. Res. Res. 22, 89S-108S. [24] Ceccherini, M.T., Ascher, J., Pietramellara, G., Vogel, T.M., Nannipieri, P., 2007. Vertical advection of extracellular DNA by water capillarity in soil columns. Soil Biol. Biochem. 39, 158–163. [25] Gulden, R.H., Lerat, S., Hart, M.M., Powell, J.R., Trevors, J.T., Pauls, K.P., Klironomos, J.N., Swanton, C.J., 2005. Quantitation of Transgenic Plant DNA in Leachate Water: Real-Time Polymerase Chain Reaction Analysis. J. Agric. Food Chem. 53, 5858–5865. [26] Pietramellara, G., Asher, J., Borgogni, F., Ceccherini, M.T., Guerri, G., Nannipieri, P. 2009. Extracellular DNA in soil and sediment: fate and ecological relevance. Biol. Fertil. Soils, 45, 219-235. [27] Paul, J.H., Jeffrey, W.H., DeFlaun, M.F., 1987. Dynamics of extracellular DNA in marine environment. Appl. Environ. Microbiol. 53, 170-179. [28] DeFlaun, M.F., Paul, J.H., Jeffrey, W.H., 1987. Distribution and molecular weight of dissolved DNA in subtropical estuarine and oceanic environments. Mar. Ecol. Progr. ser. 38, 65-73. [29] Frostegärd, A., Courtois, S., Ramisse, V., Clerc S., Bernillon, D., Le Gall F., Jeannin, P., Nesme, X., Simonet, P., 1999. Quantification of Bias Related to the Extraction of DNA Directly from Soils. Appl. Environ. Microbiol. 65, 5409-5420. [30] Lorenz, M.G., Wackernagel, W., 1994. Bacterial gene transfer by natural genetic transformation in the environment. Microbiol. Rev. 58, 563-602.

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Plant Leaf Decomposition, DNA Release, Persistence and Transfer ... 19 [31] Ogram, A., Sayler, G.S, Wackernagel, W., 1987. The extraction and purification of microbial DNA from sediments. J. Microbiol. Meth. 7, 57-66. [32] England, L.S., Pollok, J., Vincent, M., Kreutzweiser, D., Fick, W., Trevors, J.T., Holmes, S.B., 2005. Persistence of extracellular baculoviral DNA in aquatic microcosms: extraction, purification and amplification by the polymerase chain reaction (PCR). Mol. Cell. Probes, 19, 75-80. [33] Zhu, B., 2006. Degradation of plasmid and plant DNA in water microcosms monitored by natural transformation and real-time polymerase chain reaction (PCR). Water Res. 40, [34] Aardema, B. W., Lorenz, M.G., Krumbein, W. E., 1983. Protection of Sediment-Adsorbed Transforming DNA Against Enzymatic Inactivation. Appl. Environ. Microbiol. 46, 417-420. [35] Dale, P.J., Clarke, B., Fontes, E.M.G., 2002. Potential for the environmental impact of transgenic crops. Nat. Biotechnol. 20, 567-574. [36] Kögel-Knabner, I., 2002. The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter. Soil Biology and Biochemistry 34, 139–162. [37] Coûteaux, M.M., Bottner, P., Berg, B., 1995. Litter decomposition climate and litter quality. Tree 10, 63-66. [38] Monties, B., 1991. Plant cell walls as fibrous lignocellulosic composites: relations with lignin structure and function. Animal feed Science and Technology 32, 159–175. [39] Coûteaux, M.M., Bottner, P., Berg, B., 1995. Litter decomposition climate and litter quality. Tree 10, 63-66. [40] Eklind, Y., Kirchmann, H., 2000. Composting and storage of organic household waste with different litter amendments. II: nitrogen turnover and losses. Bioresource Technolology 74, 125-133. [41] Jenny, H., Gell, S.P., Bingham, F.T., 1949. Comparative study of decomposition rates of organic matter in temperate and tropical regions. Soil Science 68, 419–432. [42] Olson, J.S., 1963. Energy storage and the balance of producers and decomposers in ecological systems. Ecology 44, 322–331. [43] Wieder, R.K., Lang, G.E., 1982. A critique of the analytical methods used in examining decomposition data obtained from litter bags. Ecology 63, 1636–1642.

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[44] Widmer,F., Seidler, R.J., Watrud, L.S., 1996. Sensitive of transgenic plant marker gene persistence in soil microcosms. Mol. Ecol. 5, 603– 613. [45] Widmer, F., Seidler, R.J., Donegan, K.K., Reed, G.L., 1997. Quantification of transgenic plant marker gene persistence in the field. Mol. Ecol. 6, 1-7. [46] Gebhard, F., Smalla, K., 1999. Monitoring field releases of transgenic modified sugar beets for persistence of transgenic plant DNA and horizontal gene transfer. FEMS Microbiol. Ecol. 28, 261–272. [47] Flores, S., Saxena, D., Stotzky, G., 2005. Transgenic Bt plants decompose less in soil than non- Bt plants. Soil Biol. Biochem. 37, 10731082. [48] Nielsen, D.R., Genucheten, M.TH., Biggar, J.W., 1986. Water flow and solute transport processes in unsaturated zone. Wat. Res. Res. 22, 89S-108S. [49] Kay, E., Bertolla, F., Vogel, T.M., Simonet, P. 2002. Opportunistic Colonization of Ralstonia solanacearum-Infected Plants by Acinetobacter sp. and Its Natural Competence Development. Microb. Ecol. 43, 291-297. [50] Kay, E., Vogel, T. M., Bertolla, F., Nalin, R., Simonet, P. 2002. In situ transfer of antibiotic resistance genes from transgenic (transplastomic) tobacco plants to bacteria. Appl. Environ. Microbiol. 68, 3345-3351. [51] Rizzi, A., Pontiroli, A., Brusetti, L., Borin, S., Sorlini, C., Abruzzese, A., Sacchi, G. A., Vogel, T. M., Simonet, P., Bazzicalupo, M., Nielsen, K. M., Monier, J. M., Daffonchio, D. 2008. Strategy for in situ detection of natural transformation-based horizontal gene transfer events. Appl. Environ. Microbiol. 74, 1250-1254. [52] Bertolla, F., Pepin, R., Passelegue-Robe, E., Paget, E., Simkin, A., Nesme, X., Simonet, P. 2000. Plant Genome Complexity May Be a Factor Limiting In Situ the Transfer of Transgenic Plant Genes to the Phytopathogen Ralstonia solanacearum. Appl. Environ. Microbiol. 66, 4161-4167.

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In: Transgenic Plants Editors: Shen Y. Zhu and Jiang L.Hu

ISBN 978-1-62257-245-8 ©2012 Nova Science Publishers, Inc.

Chapter 2

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ESTABLISHMENT OF LIGHT FORMULA AND LIGHT ENVIRONMENTAL MANAGEMENT STRATEGY FOR HIGH-EFFICIENT PLANT CULTIVATION WITH ARTIFICIAL LIGHT SOURCES Wenke Liu1,2, Qichang Yang*1,2, Lingling Wei2 and Ruifeng Cheng1,2 1

Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of Agricultural Sciences, Beijing, China 2 Key Lab. of Energy Conservation and Waste Management of Agricultural Structures, Ministry of Agriculture, Beijing, China

ABSTRACT Light is a vital environmental factor that affects plant growth and development by acting on plants not only as the energy source for photosynthesis, but also as an environmental signal for photomorphogenesis. Light requirements of plants are subjected to species, cultivar, growth and developmental stages, environmental conditions and manipulation target of yield and quality. Therefore, detailed studies on light quality requirements, i.e. light fomula (LF), *

Corresponding author: Qichang Yang, Email: [email protected].

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22

Wenke Liua, Qichang Yang, Lingling Wei et al. based on biological and physiological requirement are urgently needed for getting high yield and good quality of protected plants. With the development of semiconductor solid light sources, light-emitting diodes (LEDs), detailed research on plant biology and physiology responses tomonochromatic light became possible. Therefore, the light quality requirements for all kinds of plants can be precisely and extensively studied to obtain the optimal light spectrum component for high productivity and good quality. The LF was defined as optimized monochromatic light component illuminated by LEDs for getting higher biomass and better nutritional quality of plants. Based on published literatures, red and blue light spectrum are macro-necessary LF components, and purple, green, yellow, cyan, orange and other visible light spectra are micro-beneficial LF components, while far-red and ultraviolet light spectrum is functional LF components. Generally, a kind of LF is composed of one or two kinds of necessary light spectra, several micro-beneficial and functional light spectra for special plant during a certain period and under certain environmental conditions. The LF is of crucial scientific significance that should be established for plants grown with artificial light. More importantly, the LF is an important part of light environment management strategy (LEMS). The LEMS refers to a comprehensive management method of light environment, including light intensity, LF and photoperiod for one special plant, which should be established for plants with artificial light sources. It is assumed that the LF and LEMS will be preferentially applied in plant factory with entire artificial light source.

Keywords: Light formula (LF); necessary light spectrum; light-emitting diodes (LEDs); plant factory; light environment management strategy (LEMS)

INTRODUCTION Protected vegetable cultivation has developed rapidly worldwide to meet the increasing demand for fresh vegetables. For example, it was estimated that the planting area of China of protected vegetables reached four million hectares in 2010 (Zhang, 2011). Nowadays, the protected facilities mainly include glasshouse, greenhouse, Chinese solar-greenhouse, plastic tents and plant factories for off-season vegetable cultivation. Plant factory is the top pattern of modern protected horticulture. Currently, dozens of plant factories, especially vegetable factories, are in operation in some countries, e.g. Japan, China, and Netherlands etc. As the dominant type, the vegetable factories trace

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their roots back to the Christensen Farm in Denmark, where production first started in 1957 (Takatsuji, 1986). Light is a vital environmental factor that affects plant growth and development by acting on plants not only as the sole energy source of photosynthesis (acting on chlorophyll), but also as the kind of external signal (acting on cryptochrome, phototropin and the other photoreceptors) after being intercepted and absorbed by photosynthetic tissue. It is well known that plant growth and development are regulated by light quality, light intensity and photoperiod, so above three elements are key components of light condition. Under cover, besides photosynthetically active radiation (PAR) (400-700nm), the medium-wave ultraviolet light (UV-B, 280-320nm) long-wave ultraviolet light (UV-A, 320-400nm) are important spectral components for vegetable production. However, due to the absorption and obstruction effects of cover materials (glass and plastic film), only about 88% visible light and 15.9% to 21.1% ultraviolet irradiation is transmitted into protected systems, which resulted in significantly reduction in light intensity and even substantially modification in light spectrum composition (Nitz et al., 2004; Chen and Wu, 2008; Peng and Ai, 2010). In order to obtain an optimal light environment for vegetable growth, artificial light is needed to overcome the irregular diurnal light intensity changes of natural light in greenhouse or as the sole main light source. More importantly, UV-A, UV-B and UV-C irradiation levels in closed cultivation systems with artificial light, e.g. plant factory, are very low, since ultraviolet irradiation is not present in fluorescent lamps and light-emitting diodes (400-700nm). Therefore, detailed investigation on effects of light quality of PAR and ultravoilet irradiation on growth, physiological metabolisms and nutritional quality is necessary. Generally, the improvement ofyield and nutritional quality of vegetables in protected facilities is the final target of light environment regulation. Based on current literatures, precise spatiotemporal management of light conditions (light quality, light intensity and photoperiod) can enhance yield and accumulation of health-beneficial phytochemicals in vegetables. Optimised light environment regulation can be environmentally friendly . High-quality vegetables can be produced in protected facilities. Liu et al.(2009) defined high-quality vegetables as vegetables not containing over-the-limit harmful substances (nitrate, nitrite, heavy metals and pesticides), and not having negative effects on human health after ingestion. Health-beneficial phytochemicals of vegetables are referred to some substances, such as ascorbic acid, carotenoids, phenolics, flavolds, which are influential beneficially to human health.

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In greenhouses, the artificial light source is used to support the natural light, while the artificial light source itself entirely determines the light conditions in closed vegetable factories. So, the strategy for balancing the light requirements of vegetables to the light conditions is largely different in the two kinds of protected facilities. Comparatively, the latter is relatively easy to realize. In our view, it is possible to regulate the productivity and nutritional quality of horticultural crops in protected facilities. However, what kind of light quality or wavelength combinations is suitable for a specific horticultural crop? How to manage light quality, light intensity and photoperiod synergically? What is the light environment management strategy for various protected facilities? These issues is far fully investigated, understood and answered. Therefore, more work is urgently needed to clarify the relationship between light conditions (light quality, light intensity and photoperiod) and growth & quality at every developmental stages of plants in various protected systems. Apparently, closed plant factory with artificial lighting is the ideal protected facility as objective that should be studied on above issues preferentially, and study outcome is priorily applied in closed plant factory.

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LIGHT FORMULA (LF): CONCEPT AND SIGNIFICANCE As qualitative factor, light quality is the primary component in light condition. Based on current literatures, biologically active light quality can be classified into three zones located in spectrum, including PAR zone (400nm to 700nm), far-red zone (700nm to 750nm), and ultraviolet irradiation zone (200nm t0 400nm). PAR are comprised of red, blue, green, purple, yellow, orange, cyan, while ultraviolet irradiation is comprised of UV-A (320nm to 400nm), UV-B (280nm to 320nm), and UV-C (200nm to 280nm). Higher plants are empowered with an array of photoreceptors controlling diverse responses to light quality for plant growth and its photomorphogenesis. Generally, plants have three different photoreactions: (1) red and blue light used in photosynthesis; (2) blue light used in cryptochrome and phototropin reaction systems; (3) red and far-red light enabling reversible switching of the phytochrome system (Watanabe, 2011). For natural light, light quality is often unsuitable for horticultural growth and health-beneficial phytochemical formation. Traditionally, both supplemental lighting in greenhouse and in closed vegetable factoris , usestable light conditions to maintain photosynthesis and photomorphogenesis, so light is a critical yet passive entity. With the development of light-emitting diode

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(LED), the potential to actively implement dynamic lighting strategies to control plant growth and development, and nutritional quality holds great promise in the future of protected cultivation. LEDs can tailor illumination spectra according to plant requirements. Based on the advancements in LED illumination technology, here, we put forward light formula (LF) concept for guiding the optimization of light quality application in protected plant cultivation. LF is defined as an optimized light quality component aiming at high productivity or nutritional quality under protected production. Basically, LF of plants are subjected to species, cultivar, growth and developmental stages of plant, environmental conditions and manipulation target of yield & quality. In other words, LF is cultivar-specific, dynamic and adjustable accompanied with the corresponding biological processes of plants. Not only establishment of plant LF relies on LED technology development, also application of LF relies on LED lighting systems. Surely, LED, a sort of solidstate, narrow bandwidth lighting platforms, offer a unique opportunity to realize light quality precise managements according to LF. In the future, a set of specific LF should be studied and developed for specific horticultural crops, and LF may be adjusted throughout the entire life of plant to potentially optimize traits of interest, such as morphology, yield and nutritional quality. LF and its management strategy are of great significance for closed plant factory production with artificial light. First, LF management will save energy by deleting extra light spectra; (2) LF management will maximize the yield and/or nutritional quality; (3) LF management is a basis for establish light environment management strategy (LEMS). Nowadays, LF and its management strategy is far perfectly established. Detailed studies on light fomula (LF) based on physiological requirement of plants one by one are urgently needed for high yield and quality targets.

LIGHT-EMITTING DIODES (LEDS): AN OPTIMAL TOOL FOR LF RESEARCH LF, in essence, is an optimized and integrated assembly of spectral component emitted from light sources that is suitable for plant productivity and nutritional quality formation. LED is an ideal tool to study light quality requirements of every horticultural crop. Their small size, durability, long operating lifetime, wavelength specificity, relatively cool emitting surfaces, and linear photon output with electrical input current make these solid-state

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light sources ideal for use in plant lighting designs. Because the output waveband of LEDs is much narrower than that of traditional sources of electric lighting used for plant growth, one challenge in designing an optimum light spectrum (Massa et al., 2008). LEDs have tremendous potential as supplemental or sole-source lighting systems for crop production. Several accepted advantages of LED can be classified. First, LED is energy-saving, small size and with high light efficiency; Second, LED is cold light source with low thermal radiation, so it can illuminate plants closely; Third, LED can emit multiple narrow-bandwidth monochromatic light with specific wavelengths, almost covering all biologically active light qualities. In addition, light quality biology and physiology have been gradually and extensively conducted worldwide by taking advantage of LED providing precise light spectrum and close illumination, which provide abundant data to establish LF. Totally, LED is a best candidate as light source for regulating plant biology, productivity and nutritional quality. Up to date, a number of studies and their findings are useful for selection of LED types and positioning for a variety of purposes depending on crop type and desired responses. To select specific wavelengths for a targeted plant response make LEDs more suitable for plantbased uses than many other light sources. More importantly, compound or mixed light quality according to LF can be realized through combination with various LEDs. Thus, using LED as artificial light source, light quality can be controlled with great precision as desired (Folta and Childers, 2008).

LIGHT FORMULA (LF) FOR PROTECTED HORTICULTURAL PLANTS: PRIMARY CONCLUSION LEDs have become an optimal tool for LF research of plants, particularly vegetables in closed plant factory or growth chamber since 1990s last century. It is well known that NASA’s Kennedy Space Center and Purdue University had made great contributions for LED lighting technology (Folta and Childers, 2008; Massa et al., 2008). Today, scientists of many countries, e.g. Japan, USA and China, are positively working in this field. With rapid development of LED technology in 21th century, research on light biology and physiology of vegetables accelerated because more pure light wavelengths were emitted from novel LED lighting systems. In 1990s, some initial studies focused on usefulness of LEDs as a sole source or as supplemental lighting for plant growth. For PAR, biological

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effects of red light plus blue light were extensively investigated on lettuce (Bula et al., 1991; Hoenmecke et al., 1992; Yanagi et al., 1996b; Yorio et al., 1998), strawberry (Yanagi et al., 1996a) and pepper (Brown et al., 1995), wheat (Goins et al., 1997) and rice (Matsuda et al., 2004). Goins et al. (1997) indicated that wheat can complete its life cycle under red LEDs alone, but larger plants and greater amounts of seed are produced in the presence of red LEDs supplemented with a quantity of blue light. Matsuda et al. (2004) also found that rice plants grown under a combination of red (660 nm) and blue (470 nm) LEDs sustained higher leaf photosynthetic rates than did leaves from plants grown under red LEDs only. Yorio et al. (1998) summarized previous blue light work and reported that yield of lettuce, spinach, and radish crops grown under red LEDs alone was reduced compared with when blue fluorescence was included to give the same final PPF. Schuerger et al. (1997) examined changes in leaf anatomy of pepper under different color combinations of light. They used red (660 nm) LEDs combined either with FR (735 nm) LEDs or BF lamps at the same PPF. Their results indicated that leaf thickness and number of chloroplasts per cell depended much more on the level of blue light than the red:FR ratio. Many horticultural and grain crops had been cultivated successfully under LED light sources with red light, blue light or red plus blue light quality, including lettuce, spinach, strawberry, tomato, cucumber, potato, wheat and rice as white light. Furthermore, mixed light quality comprised of suitable red light and blue light ratio did facilitate high productivity or nutritional quality, e.g. lettuce (Zhou et al., 2011), spinach (Ohashi-Kaneko et al., 2007), strawberry (Yanagi et al., 2006). Previous work has focused on the proportion of blue light required for normal plant growth as well as the optimum wavelength of red and the red/far-red ratio. Thus it can be concluded that, as white light, blue light and red light is necessary light quality for plant cultivation. Combination of red and blue lighting was an effective light source for several crops. Other light qualities were also evidenced in yield or quality benefits in vegetable production under artificial lighting. Kim et al. (2004) found that the addition of 24% green light to red and blue LEDs enhanced lettuce growth since green light can better penetrate the plant canopy and potentially increase plant growth by increasing photosynthesis. The addition of green light could offer more benefits, since green light can better penetrate the plant canopy and potentially increase plant growth by increasing photosynthesis from the lower canopy leaves. Similarly, other light wavelengths , such as yellow, orange, purple, cyan and so on, may affect horticultural crops to some extent under certain conditions. This needs further investigations. Liu et al. (2011) found

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that cherry tomato seedlings were significantly stronger and shorter under ?, than under yellow, green and red were weaker and higher. Photosynthetic pigments were shown to have significant difference under respective light irradiations of LEDs. Taken together, red plus blue and red plus blue and green light of LEDs were shown to be beneficial factors for the growth and photosynthesis of cherry tomato seedlings. Recently, usefulness of supplemental ultravoilet radiation on growth and nutritional quality of protected vegetables were also explored. Using UV-A, blue, green, red, and far-red LEDs, authors investigated the effects of different supplemental light qualities on phytochemicals and growth of baby leaf lettuce grown white fluorescent lamps as the main light source inside a growth chamber (Li and Kubota, 2008). Briefly, supplemental blue or UV-A could enhance accumulation of anthocyanins, supplemental blue also increased carotenoids concentration, supplemental R could increase phenolics concentration while supplemental far-red light could increase biomass, but result in lower phytochemical concentrations. A controlled light quality with an appropriate ratio of blue, red or far-red light quality provided as supplemental light may improve phytochemical content and biomass of plants grown under white light. Further studies are needed to describe the effects of different ratios of selected light qualities in both growth chamber and greenhouse conditions. The results demonstrated that supplemental light quality could be strategically used to enhance nutritional value and growth of baby leaf lettuce grown under white light (Li and Kubota, 2008). Via continuous lighting by LED, the contribution of red light to significant βcarotene expression and antioxidant activity for nutrition and health benefits are emphasized (Wu et al., 2007). Also, the potential for increasing secondary compounds in vegetables by using supplemental selected UV irradiation have been extensively investigated (Tsormpatsidis et al., 2008; Voipio and Autio, 1995). UV radiation can be regarded as a stress factor which is capable of significantly affecting plant growth characteristics. Generally, plant height, leaf area, leaf length have been showed to decrease, whereas leaf thickness was increased in response to UV-B radiation (Rozema et al., 1997). Plants produce a wide range of flavonoids and related phenolic compounds which tend to accumulate in leaves of higher plants in response to UV radiation (Rozema et al., 1997). Plants may produce secondary products to protect them against UV light damage, but these metabolites also play an important role in human health. Phenolics, flavonoids and anthocyanins are responsible for antioxidant activity in fruits and vegetables (Cao et al., 1997).

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Based on published literatures, red, blue, and compound white light are macro-necessary light spectrum, and purple, green, yellow and orange light are micro-beneficial light spectrum, while far-red light and ultravoilet light are beneficial light spectrum. Spectral changes of illumination evoked ifferent morphogenetic and photosynthetic responses, which can vary among different plant species. However, other light spectrum except photosynthetic active radiation, far-red and ultravoilet light is unvalid for plant cultivation. Our hypothesis is that one or two kind of necessary light quality, special microbeneficial and beneficial light quality can make up a light formula for a kind of plant during certain period and certain environmental conditions. LF is a crucial scientific issue that should be established for protected cultivation plants with artificial light or plants that needed supplemental light.

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LF: AN IMPORTANT PART OF THE LIGHT ENVIRONMENT MANAGEMENT STRATEGY (LEMS) Establishment of LF will lay a foundation for establishing light environment management strategy (LEMS) for given protected plant species cultivated with artificial light source. Besides LF, light intensity and photoperiod are key contents of the light environment management. The LEMS refers to a comprehensive management method of light environment, including LF, light intensity and photoperiod for entire life of one special plant species, which should be established to facilitate high productivity and good nutritional quality with artificial LED light sources. By using LED lighting systems, it is possible to continually adjust fluence rate, wavelength combinations, and photoperiods to actively manipulate plant morphology and production, rather than using a stable light condition traditionally. The application of LEMS will be used in the plant factory for the advantages of entire artificial light source, cultivation with nutrient solution and intelligent environmental control. Furthermore, vegetable production, particularly leafy vegetable cutivation, will benefit preferentially from the use of LEMS in plant factory. Corresponding LED lighting systems for plant factory should be designed and developed. Ideally, for an optimum plant production and product quality, light environment have to be adapted to the needs of the plants at every moment controlled by LEMS.

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CONCLUSION With the development of LED illumination technology, light biology and physiology of protected horticultural crops were conducted extensively. Simultaneously, LEDs are bringing a breakthrough in artificial lighting in protected horticulture. LEDs provide a tool to facilitate the study and establishment of the LF and LEMS of every target horticultural crops. In return, LF will provide technique parameters for design of species-specific lighting regimes may help maximize plant productivity and benefits of food quality. Optimization of lighting system, establishement of LF and LEMS will drive plant cultivation in plant factory greatly either in productivity or nurtritional quality.

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ACKNOWLEDGMENTS This study was supported by the National High Technology Research and Development Plan of China（863 Project, grant No. 2011AA03A114) and the Basic Scientific Research Fund of National Nonprofit Institutes (BSRF201004) and 2012-2013, Institute of Environment and Sustainable Development in Agriculture, CAAS.

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Folta K.M., Childers K.S. 2008. Light as a growth regulator: controlling plant biology with narrow-bandwidth solid-state lighting systems. HortScience. 43: 1957-1963. Goins, G.D., N.C. Yorio, M.M. Sanwo, and C.S. Brown. 1997. Photomorphogenesis, photosynthesis, and seed yield of wheat plants grown under red light-emitting diodes (LEDs) with and without supplemental blue lighting. J. Expt. Bot. 48:1407–1413. Hemming S. 2011. Use of natural and artificial light in horticulture-interaction of plant and technology. Acta Horticulturae , 907: 25-35. Kim H.H., Wheeler R.M., Sager J.C., Goins G.D. 2004. A comparison of growth and photosynthetic characteristics of lettuce grown under red and blue light-emitting diodes (leds) with and without supplemental green LEDs. Acta Hort. 659:467-475. Li .J.C., Liu W.K., Yang Q.C. 2010. Strategic idea of replacing resources with environmental factors in agricultural production through protected agricultural technology. Chinese Agricultural Science Bulletin 26(3):283285. Liu W.K., Yang Q.C., Du L. F. 2009. Soilless cultivation for high-quality vegetables with biogas manure in China: feasibility and benefit analysis. Renewable Agriculture and Food Systems. 24(4):300–307. Liu X.Y., Chang T.T., Guo S.R., Xu Z.G., Li J. 2011. Effect of different light quality of led on growth and photosynthetic character in cherry tomato seedling. Acta Hort.907: 325-330. Liu XiaoYing, Guo ShiRong, Xu Zhiang , Jiao XueLei，Takafumi Tezuka. 2011Regulation of chloroplast ultrastructure, cross-section anatomy of leaves and morphology of stomata of cherry tomato by different light irradiations of LEDs. Hortiscience.45 (2):1-5. Marcelis L.F.M., Snel J.F.H., de Visser P.H.B., et al. 2006. Quantification of the growth responses to light quantity of greenhouse grown crops. Acta Horticulturae711:97-104. Massa G. D., Kim H.-H., Wheeler R. M., Mitchell C. A. 2008. Plant productivity in response to LED lighting. HortScience. 43(7): 1951–1 956. Matsuda, R., K. Ohashi-Kaneko, K. Fujiwara, E. Goto, and K. Kurata. 2004. Photosynthetic characteristics of rice leaves grown under red light with or without supplemental blue light. Plant & Cell Physiol. 45:1870–1874. Matsuda, R., K. Ohashi-Kaneko, K. Fujiwara, E. Goto, and K. Kurata. 2004. Photosynthetic characteristics of rice leaves grown under red light with or without supplemental blue light. Plant & Cell Physiol. 45:1870–1874. Morrow, R.C. 2008. LED lighting in horticulture. HortScience 43:1947–1950.

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Nitz, G. M., and Grubmuller, E., and Schnitzler,W. H. Differential flavoniod response to PAR and UV-B light in chive (Allium schoenoprasum L.). Acta Hort. , 2004, 659: 825–830. Ohashi-Kaneko, K., Takase, M., Kon, N., Fujiwara, K., Kurata, K., 2007. Effect of light quality on growth and vegetable quality in leaf lettuce, spinach and komatsuna. Environ. Control Biol. 45, 189–198. Peng Y, Ai X. A review on effects of UV-B increase on vegetables. Modern Horticulture, 2010，6：16-17. Rozema, J., Staaij, J.vd., Bjorn, L.O., Caldwell, M., 1997. UV-B as an environmental factor in plant life: stress and regulation. Trends Ecol. Evol. 12, 22–28. Samuolienė G., Brazaitytė A., Urbonavičiūtė A., Šabajevienė G., Duchovskis P. The effect of red and blue ligjht component on the growth and development of frigo strawberries. Zemdirbyste-Agriculture, 2010,97(2): 99-104. Schuerger, A.C., C.S. Brown, and E.C. Stryjewski. 1997. Anatomical features of pepper plants (Capsicum annuum L.) grown under red light-emitting diodes supplemented with blue or far-red light. Ann. Bot. (Lond.) 79:273– 282. Takatsuji M. Vegetable plant factory. Maruzen, Tokyo, 1986. Tsormpatsidis, E., Henbest, R.G.C., Davis, F.J., Battey, N.H., Hadley, P., Wagstaffe, A., 2008. UV irradiance as a major influence on growth, development and secondary products of commercial importance in Lollo Rosso lettuce ‘Revolution’ grown under polyethylene films. Environ. Exp. Bot. 63, 232–239. Wang Y.L., Wang X.L., Yu E.M. Effects of supplementary radiation of UV-B and red light on fruit quality of tomato in winter plastic greenhouse. Acta Bot. Boreal. -Occident. Sin, 2000, 20(4): 590-595. Watanabe H. Light-controlled plant cultivation system in Japan-development of a vegetable factory using LEDs as a light source for plants. Acta Horticulturae, 2011, 907:37-44. Wu M C, Hou C Y, Jiang C M, Wang Y T, Wang C Y,Chen H H,Chang H M. 2007.A novel approach of LED light radiation improves the antioxidant activity of pea seedlings. Food Chemistry, 101（4）:1753-1758. Yanagi, T., K. Okamoto, and S. Takita. 1996a. Effect of blue and red light intensity on photosynthetic rate of strawberry leaves. Acta Hort. 440:371– 376.

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Yanagi, T., K. Okamoto, and S. Takita. 1996b. Effect of blue, red, and blue/red lights of two different PPF levels on growth and morphogenesis of lettuce plants. Acta Hort. 440:117–122. Yanagi, T., T. Yachi, N. Okuda, and K. Okamoto. Light quality of continuous illuminating at night to induce floral initiation of Fragaria chiloensis L. CHI-24-1. Sci. Hort., 2006,109:309–314. Yorio, N.C., G.D. Goins, H.R. Kagie, R.M. Wheeler, and J.C. Sager. 2001. Improving spinach, radish, and lettuce growth under red light-emitting diodes (LEDs) with blue light supplementation. HortScience 36:380–383. Zhang Z.B. Development countermeasure of protected vegetables with low carbon production technology. In: Yang Q.C., Kozai T., Bot G.P.A. Protected Horticulture Advances and Innovations-Proceedings of 2011 the 2nd High-level International Forum on Protected Horticulture (Shouguang·China). China Agricultural Science and Technology Press. Beijing. 2011. 9-13.

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In: Transgenic Plants Editors: Shen Y. Zhu and Jiang L.Hu

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Chapter 3

HARVEST-INDUCIBLE GENES AND PROMOTERS IN ALFALFA Jian Zhang and Larry R. Erickson

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Plant Agriculture Department, University of Guelph, Guelph ON, Canada

ABSTRACT The harvesting and storing of alfalfa is a routine practice in the agricultural industry worldwide. A study of the physiological and biochemical changes that occur after harvesting may help to understand how plants respond to this process. To investigate gene expression in harvested alfalfa (Medicago sativa), cDNA from non-harvested and harvested plants in the field was subjected to subtractive hybridization to identify, in particular, those genes that are induced by the harvesting treatment. Three different genes, named, hi7, hi11 and hi12, were isolated and analysed. Northern blot analysis confirmed that hi7, hi11 and hi12 are strongly induced during a post harvest incubation period. The promoters of these genes were isolated and characterized. Reporter gene driven by the promoter has been transformed into transgenic plants. The hi12 promoter showed it is harvesting inducible.

Keywords: Harvest-inducible gene, promoter, transformation, Alfalfa 

Current address: Alberta Innovates-Technology Futures, Vegreville, AB, T9C1T4 Canada.

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INTRODUCTION Alfalfa (Medicago sativa L.) is probably native to Asia Minor and the Caucasus mountain area, and has been cultivated since antiquity. Alfalfa was likely cultivated before recorded history in a region spanning from China in the east to Spain in the west and from as far north as Sweden to as far south as North Africa. Recently, it has become an important crop in South Africa, Australia, New Zealand and North and South America [1]. Alfalfa is the most important forage legume crop in North America. In Ontario, forages were grown on about 2.5 million ha with a value of hundreds of millions of dollars of commercial hay. A major component of the forage crop in Ontario is alfalfa. Alfalfa is highly regarded for its feeding value which is due to its high protein content, and it is often called the “Queen of Forages” [2]. Alfalfa is harvested and stored primarily as hay or silage for use on farm. In all cases, the above ground part of the plant is cut near the base and laid in a swath in the field to wilt and reduce the water content, a process that can last for a period ranging from a few hours to a few days depending on weather conditions. While lying in the swath, the harvested alfalfa plant tissue continues to function metabolically and the synthesis of mRNA and protein continues at least until the moisture level declines to ~20%. Ferullo et al. have demonstrated that proteins are synthesized in harvested alfalfa plants, which are specific to harvesting conditions, and are not induced by wounding or water stress treatments on non-harvested plants [3]. However, the identity of these proteins was not determined and the biochemical and physiological aspects of protein metabolism after harvesting are largely unknown. Subtractive hybridization is a powerful method allowing one to obtain cDNA libraries enriched with transcripts that are present in one tissue or individual organism but not in another [4]. Straus and Ausubel were probably the first to report this method [5], and since then, subtractive hybridization has been found research applications in many disciplines ranging from identification of methylation changes during tumourigenesis [6] to isolation of diagnostic probes for infectious diseases [7]. In plants, it has been used to study the role of Chk1-a signaling element in the development of fungal pathogens on the plant host [8]. Matvienko et al. used a normalized, subtractive cDNA library enriched for transcripts differentially abundant in Triphysaria versicolor root tips treated with the allelopathic quinone 2,6-dimethoxybenzoquinone (DMBQ) to study the quinone detoxification mechanism in plants [9]. Zhang et al. constructed a cotton (Gossypium arboreum L.) drought-related cDNA library using using

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subtractive hybridization [10]. Salt-induced genes in Suaeda maritima were cloned using PCR-based suppression subtractive hybridization [11]. The expression of a foreign protein in a transgenic plant may be somewhat toxic or inhibitory to growth and development. The consequence of expressing such proteins with a constitutive promoter may range from regeneration of plants with a low concentration of that protein to distorted plant phenotype and sterility. A practical solution to this problem is to control expression of these proteins by inducible promoters, an approach well established in microbial systems. An objective of the research conducted for this research was to investigate gene expression in harvested alfalfa tissue and specifically to isolate the genes that are induced by harvesting. Isolation and characterization of these genes and their regulatory elements could provide new information on the function of such genes in plants, as well as provide a novel system for expressing foreign proteins in harvested plant tissue. Since induction would occur only after harvesting, there would be no negative effect on plant growth and development due to the constitutive expression of foreign proteins.

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MATERIALS AND METHODS Plant Materials Alfalfa plants, cv Gala (Northrup King, Canada) were harvested from a field in the second year of growth on a farm just south of Guelph, Ontario in sunny conditions and an air temperature around 25-28oC, just before noon. Plants were harvested from a random area in the field of approximately 1 m2. The harvested whole plant samples were laid flat on the remaining stubble to wilt for approximately 1 h and reduce moisture content, as is done in normal harvest operations. The harvested samples taken from 10 plants were then covered with aluminium foil and stored at room temperature for 30 min, 2 h, 6 h and 24 h before RNA was extracted from leaves and petioles. The nonharvested control plants were immediately frozen in liquid nitrogen.

RNA Extraction Total RNA was isolated from control samples and harvested samples using TrizolTM Reagent (Gibco BRL). The quality and quantity of total RNA

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were determined by formaldehyde-denatured agarose gel electrophoresis and spectroscopy. A QIAGene mRNA isolation kit was used to isolate poly A+ mRNA from 1 ug total RNA from harvested tissue. The mRNA from nonharvested tissues was designated as the driver and the combined mRNA from tissue extracted 30 min and 6 h post-harvest was designated as the tester.

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Subtractive Hybridization A CLONTECH PCR-SelectTM cDNA Subtraction Kit (CLONTECH Laboratories, Inc. Catalog # K1804) was used to generate a harvest-inducible subtractive cDNA library, according to the manufacturer’s instructions. Tester and driver double stranded (ds) cDNAs were generated from both the harvestinduced mRNA population and the non-harvest induced mRNA population. Tester and driver ds cDNAs were then digested separately with restriction endonuclease RsaI to obtain short, blunt-ended molecules in the following typical reaction: 43.5 μl ds cDNA, 5.0 μl 10× Rsa I restriction buffer and 1.5 μl Rsa I (10 units/μl). The reaction was mixed by a brief vortexing and centrifugation, followed by incubation at 37 ºC for 2 h. The reactions were analyzed by agarose gel electrophoresis for efficiency of digestion and terminated by adding 2.5 μl of a 20 mM EDTA/glycogen mix and purified by a standard phenol/ chloroform/iosamyl alcohol procedure. The 50 μl reaction was divided equally into two 25 μl tester populations, each ligated with different adaptors, adaptor1 and adaptor 2r. However, no adaptor was ligated to the driver cDNA. Subtractive hybridization of the driver cDNA and adaptor 1 ligated tester cDNA and adaptor 2r ligated cDNA led to equalization and enrichment of differentially expressed sequences and generated two primary hybridization populations. The reaction was carried out as follows: 1.5 μl driver cDNA, 1.5 μl adaptor-1 ligated tester cDNA denatured at 98 ºC for 1.5 min and incubated at 68ºC for 8 h. The second hybridization was conducted between these two populations without denaturing. The remaining equalized and subtracted ss tester cDNAs formed new hybridized molecules that were present only in the harvestinduced population. A PCR was used to obtain the desired harvest-inducible cDNA fragments. In the PCR reactions, primers specific to the adaptors were used and each reaction was comprised of 3 μl cDNA, 0.2mM dNTP mixture, 1.5 mM MgCl2, 0.2 μM of each primer and 2 Units of Taq DNA polymerase (BRL Life Technologies). The PCR program was as follows: (1) 94 C 3 min;

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(2) 35 cycles of: 94 C for 40 sec, 66 C for 1 min, 72 C for 1 min, (3) 72 C for 10 min, and (4) stationary at 4 C.

Cloning of Harvest-Inducible cDNA Fragments The PCR products were cloned into TOPOTM TA cloning vector (InvitroGen, Canada) to generate the harvest-inducible cDNA library. The transformation was conducted according to the protocol provided with the TOPOTM TA Cloning kit®. The plates were incubated overnight at 37 ºC and the white colonies were chosen for further analysis. Purified plasmid DNA, extracted by Genelute plasmid mini-pre kit (Sigma-Aldrich, St. Louis, Mo), was submitted for sequencing to Laboratory Services, a division of the University of Guelph. The PCR screening of bacteria, plasmid DNA purification, and sequencing procedures were carried out according to standard DNA manipulation methods [12].

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Northern Blot Analysis Northern blot analysis of harvest-inducible transcripts was conducted with probes excised from hi11 and hi12 cDNA clones using Rsa I. Total RNA was isolated from alfalfa plants following a post-harvest period of 0, 45 min, 2 h, 6 h and 24 h. Approximately 10 μg total RNA was electrophoresed in a formaldehyde denaturing 1 % agarose RNA gel. Blotting, hybrization and washing conditions were according to the manufacturer’s instruction manual (GeneScreen, DuPont). A α-32P isotope-labelled probe was hybridized with the membrane in 5X SSC, 10X Denhardt’s solution, 0.1 % sodium pyrophosphate, 50 % formamide, 10 % dextran sulfate, 0.2 % SDS and 100 μg/mL denatured ss DNA at 42 ˚C for 24 h.

Isolation Promoter of the hi12 Gene by Genomic Walking A CLONTECH Universal GenomeWalker kit (CLONTECH Laborotory Inc.) was used for isolating the upstream region of the hi12 gene according to the manufacturer’s instructions. Five genomic walking libraries were generated from the genomic DNA digested with five restriction enzymes. The hi12 gene specific primer h12-98C (5'-CTTGTGACCAACTTCTTGATG

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TCTTCC-3') was designed based on cDNA sequence data from Inoue et al (1998). The primary PCR reaction was conducted as follows: 7 cycles: 94ºC for 25 s, 72ºC for 3 min; 32 cycles: 94ºC for 25 s, 67ºC for 3 min and an additional final step of 67ºC for 7 min. The secondary touchdown PCR with a nested primer provided by the kit was performed according to the manufacturer’s instructions. The PCR was performed with the following cycle program: 5 cycles: 94ºC for 25 s, 72ºC for 3 min; 20 cycles: 94ºC for 25 s, 67ºC for 3 min and an additional final step of 67ºC for 7 min. The PCR screening of bacteria, plasmid DNA purification, and sequencing procedures were carried out according to standard DNA manipulation methods.

Figure 1. The sequences of hi1, hi8, hi11 and hi12 cDNAs.

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RESULTS Isolation of Harvest-Inducible cDNA Clones

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A subtractive cDNA library was obtained from the hybridization of noharvest and 6 h post-harvest induced mRNA populations (Figure 1). The library contained 12 colonies, from which four unique cDNA sequences were identified, hi1, hi8, hi11 and hi12 (hi-harvest-inducible. The hi1 cDNA fragment of 551 bp in length, has high homology to Arabidopsis rubisco activase (Rca1) mRNA (AY130300) (Figure 2 a). Rubisco activase is a stromal protein that regulates the activation of rubisco, the most abundant enzyme in plants [13-14]. The hi8 cDNA fragment was 174 bp in length and has high homology to the phosphoribulokinase precursor mRNA in common ice plant (Mesembryanthemum crystallinum), an enzyme which may play an important role in the regulation of the Calvin cycle in higher plants [15] (Figure 2 b).

Figure 2. Secondary PCR amplification of harvest-inducible cDNA subtractive library. lane1: DNA ladder Фx174/Hind III; lane 2: PCR products amplified from subtractive hybridization between mRNA populations of non-harvested and harvested tissue at 30 min. lanes 3, 4: PCR products amplified from subtractive hybridization between mRNA populations from non-harvested and harvested tissue at 6 h.

The hi11 cDNA was 357 bp in length and has high homology to a putative protein region with no known function in the genome database of Arabidopsis thaliana (Figure 2 c). The hi12 cDNA was 348 bp long and was identical to the 3’ end of the mRNA of Medicago sativa S-adenosyl-L-methionine: transcaffeoyl-CoA 3-O-methyltransferase (CCOMT) mRNA [16-17], which catalyzes the formation of mono- or dimethoxylated lignin precursors (Figure 2 d).

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The activity of this enzyme has been shown to increase from the first to the sixth internode in elongating stems of alfalfa and precedes the deposition of lignin.

Northern Blot Analysis

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Northern analyses of hi11 and hi12 expression in harvested alfalfa tissue showed no detectable mRNA prior to harvest and a significant increase in transcript levels with an increase in the length of the post-harvesting incubation period. The harvesting treatment strongly induced hi11 at 30 min, reaching a peak between 6 and 24 h after harvesting. The steady state level of hi12 mRNA also showed a significant increase until 24 h post harvest, which was the longest post-harvest incubation period evaluated (Figure 3).

Figure 3. Northern blot analysis of hi11 and hi12 transcript levels in alfalfa leaf tissue following different post-harvest incubation periods. Arrows at left indicate expected size of mRNA. Approximately 10 μg RNA was loaded per lane. lane 1: 0 min; lane 2: 30 min; lane 3: 2 h; lane 4: 6 h; lane 5: 24h..

GUS Gene Construct with the hi12 Gene Promoter and Plant Transformation A hi-12 Bgl II primer (5'-AGATCT-GATATGTGTTGAAGAGTTGA-3'), and hi12 Sac I primer (5'-GAGCTCGAATTCTTAAGTTACGTGA-3') were designed for amplification of the hi12 promoter region. The GUS gene construct containing the hi12 promoter was derived from pCAMBIA binary vectors. A 860 bp long hi12 promoter fragment (5 to -854) was used for the

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hi12: GUS constructs [24]. The hi12 promoter driven construct were mobilized into Agrobacterium tumefaciens strain C58, and tobacco, cv Pet H4, leaf discs were infected with bacterial cultures containing the individual constructs. The infected leaf discs were incubated at 28ºC, in the dark for 4 days, and then transferred to antibiotic selection MS medium, containing 25 mg/l hygromycin or 100 mg/l kanamycin, at 24C with a 16-h light/8-h dark cycle until shoots appeared.. The shoots were excised from callus tissue and maintained in vitro on one-half MS medium at 24C with a 16-h light/8-h dark cycle, and then transplanted to the greenhouse. Histochemical staining for GUS activity was performed essentially as described by Jefferson et al. [23].

GUS Gene Activity Driven by hi12 Promoter

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The hi12 promoter (hi12-1301 construct) provided strong activity in transgenic tobacco and Medicago truncatula plants when the plant tissues had been subjected to a post harvest incubation period of 24 h (Figure 4).

Figure 4. Histo-chemical staining for β-glucuronidase (GUS) activity in transgenic tobacco and M. truncatula leaf tissue, which containing the hi12 promoter-driven construct following a post-harvest incubation period of 0h-0 hour and 24h-24 hours.

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DISCUSSION An underlying hypothesis of this research was that the expression of some genes is up-regulated in harvested alfalfa tissue, and an objective was to isolate and characterize some of these genes. cDNA clones of harvestinducible genes in alfalfa were isolated using PCR-based subtractive hybridization, a technique used to identify genes that are expressed in one mRNA population but not another [4]. In this process, all cDNAs that were not induced by harvesting were eliminated by hybridization. The subtracted cDNA library obtained by hybridization of cDNA populations generated from nonharvested and harvested alfalfa tissue contained several genes that were induced by the harvesting treatment. This was demonstrated by the Northern blot analysis, in which transcript levels of the genes showed a significant increase during a 6-24 h post-harvest incubation period compared to low levels in non-harvested alfalfa tissue. These results support the hypothesis that there are harvest-inducible or harvest-associated genes in the alfalfa plant. There were 12 clones generated from subtractive hybridization; this relatively small number of unique clones could be due the low number of genes involved in the harvesting response in alfalfa. The efficiency and specificity of the hybridization and cloning procedures, however, were verified in a parallel control experiment provided by the Clonetech kit with human skeletal mRNA, where a single clone was isolated. Moreover, the small number of clones is consistent with the small number of proteins (11) that Ferullo et al. found to be specific to the harvesting response [3]. The harvest-inducible cDNA clones, which were identified in this study, showed a broad range of homologies, and could be divided into three classes: response to light, general response to stress and others. Based on their homologies, hi1, and hi8 are possibly involved in light response. hi1 is identical to the alfalfa rubisco activase (RCA, AAN15946), but not yet fully characterized. Rubisco activase is an ATP-dependent enzyme, which appears to play a key role in photosynthesis [18-19]. Consequently, the enzyme is abundant in photosynthetic tissue, and probably accounts for 2 % of total soluble protein from spinach leaves [20]. Although it is evident that hi1 responds to the harvesting treatment, the function of this enzyme in such tissue has yet to be determined. It has been suggested, however, that RCA is involved in the activation of rubisco through carbamylation. Conceivably increased RCA availability in harvested tissue may reflect a general metabolic shift from photosynthesis to a more catabolic process of remobilization of amino acids regenerated from the photosynthetic apparatus. Based on

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homologies hi12 would appear to be associated with one or more general stress response functions in plants. hi12 is the gene for the S-adensoyl-Lmethionine-trans-caffeyol-CoA-O- methytransferase (CCOMAT) in alfalfa, an enzyme known to be involved in lignin synthesis in stems and implicated in mounting a physical defence to pathogen infection [21-22]. hi11 cannot yet be associated with any known function on the basis of homology. The use of harvest-inducible promoters in alfalfa may provide a unique and powerful inducible expression system in transgenic plants. Such a system would provide a measure of biological containment, as the foreign proteins are not produced in the transgenic plant until the application of the harvesting treatment, at which time the plant tissue is collected and stored. In addition, the crop can be harvested and stored by using conventional farm equipment and facilities. However, a much more complete analysis of transgenic plants containing foreign genes controlled by hi promoters is needed to verify the utility of such s system. The basal level of expression of reporter gene, such as GUS, needs to be determined in a range of tissue and under a range of conditions, such as drought, heat, cold and wounding by insects or pathogen.

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ACKNOWLEDGMENTS Extensive support for this research was provided by the Natural Sciences and Engineering Research Council of Canada, Strategic Grants Program.

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D.A. Samac, S. Austin-Phillips, Alfalfa (Medicago sativa L.). Methods Mol. Biol. 343 (2006) 301-311. D.K. Barnes, B.P. Goplen, J.E. Baylor, “Highlights in the USA and Canada” in Alfalfa and Alfalfa Improvement. eds, American Society of Agronomy, Inc. Madison, 1988, pp1-22. J. Ferullo, L. Verzina, Y. Castonguay, G. Allard, P. Madeau, C. Willemot, S. Laberge, Post-harvest alteration of in vitro translatable mRNA population in alfalfa (Medicago. sativa L.). Crop Sci. 36 (1996) 1011-1016. B. Blumberg, J.C. Belmonte, Subtractive hybridization and construction of cDNA libraries. Methods Mol. Biol. 461 (2008) 569-587.

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Jian Zhang and Larry R. Erickson D. Straus, F.M. Ausubel, Genomic subtraction for cloning DNA corresponding to deletion mutations. Proc. Natl. Acad. Sci. USA 87 (1990) 1889-1893. G.J. Brock, T.H. Huang, C.M. Chen, K.J. Johnson, A novel technique for the identification of CpG islands exhibiting altered methylation patterns. Nucleic. Acids Res. 29 (2001) e123. Y.N. Parsons, S. Panagea, C.M. Smart, M.J. Walshaw, A. Hart, C. Winstanley, Use of subtractive hybridization to identify a diagnostic probe for a cystic fibrosis epidemic strain of Pseudomonas aeruginosa. J. Clin. Microbiol. 40 (2002) 4607-4611. S. Lev, B.A. Horwitz, A miotogen-activated protein kinase pathway modulates the expression of two cellulose genes in Cochliobolus heterostrophus during plant infection. Plant Cell 15 (2003) 835-844. M. Matvienko, M.J. Torres, J.I. Yoder, Transcriptional responses in the hemiparasitic plant Triphysaria versicolor to host plant signals. Plant Physiol. 127 (2001) 272-282. L. Zhang, F.G. Li, C.L. Liu, C.J. Zhang, X.Y. Zhang, Construction and analysis of cotton (Gossypium arboreum L.) drought-related cDNA library. BMC Res. Notes 2 (2009) 120. B.B. Sahu, B.P. Shaw, Isolation, identification and expression analysis of salt-induced genes in Suaeda maritima, a natural halophyte, using PCR-based suppression subtractive hybridization. BMC Plant Biol. 9 (2009) 69. J. Sambrook, D.W. Russell, Molecular Cloning; A laboratory Manual, Cold Spring Harbor laboratory Press, New York, 2001. N. Zhang, R.P. Kallis, R.G. Ewy, A.R. Portis Jr., From the cover: light modulation of Rubisco in Arabidopsis requires a capacity for redox regulation of the larger rubisco activase isoform. Proc. Natl. Acad. Sci. USA 99 (2002) 3330-3304. R.F. Sage, D.A. Way, D.S. Kubien, Rubisco, Rubisco activase, and global climate change. J. Exp. Bot. 59 (2008) 1581-1595. N. Wedel, J. Soll, B.K. Paap, CP12 provides a new mode of light regulation of Calvin cycle activity in higher plants. Proc. Natl. Acad. Sci. USA 94 (1997) 10479-10484. K. Inoue, V.J. Sewalt, G.B. Murray, W. Ni, C. Sturzer, R.A. Dixon, Developmental expression and substrate specificities of alfalfa caffeic acid 3-O-methyltransferase and caffeoyl coenzyme A 3-Omethyltransferase in relation to lignification. Plant Physiol. 117 (1998) 761-770.

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[17] Q. Yang, H.X. Trinh, S. Imai, A. Ishihara, L. Zhang, H. Nakayashiki, Y. Tosa, S. Mayama, Analysis of the involvement of hydroxyanthranilate hydroxycinnamoyltransferase and caffeoyl-CoA 3-O-methyltransferase in phytoalexin biosynthesis in oat. Mol. Plant Microbe. Interact. 17 (2004) 81-89. [18] M.E. Salvucci, W.L. Ogren, The mechanism of rubisco activase: insights from studies of properties and structure of the enzyme. Photosynth. Res. 47 (1996) 1-11. [19] S. Eberhard, G. Finazzi, F.A. Wollman, The dynamics of photosynthesis. Annu. Rev. Genet. 42(2008) 463-515. [20] S.P. Robinson, V.J. Streusand, J.M. Chatfield, A.R. Portis Jr., Purification and assay of rubisco activase from leaves. Plant Physiol. 88 (1988) 1008-1014. [21] R.A. Dixon, N.L. Paiva, Stress-induced phenylpropanoid metabolism. Plant Cell 7 (1995) 1085–1097. [22] J.L. Ferrer, M.B. Austin, C. Stewart Jr., J.P. Noel, Structure and function of enzymes involved in the biosynthesis of phenylpropanoids. Plant Physiol. Biochem. 46 (2008) 356-370. [23] RA Jefferson, TA Kavanagh, MW Bevan, GUS fusions: betaglucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. (1987) 6:3901-3907. [24] J. Zhang, L.R. Erickson, Harvest-inducibility of the promoter of alfalfaS-adenosyl-Lmethionine: trans-caffeoyl-CoA3-O-methyltransferase gene. Mol. Biol. Rep. (2012) 39:2489–2495.

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In: Transgenic Plants Editors: Shen Y. Zhu and Jiang L.Hu

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Chapter 4

TOXIC IMPACTS OF THREE VETERINARY ANTIBIOTICS ON SEED GERMINATION AND GROWTH AS WELL AS NUTRITIONAL QUALITY OF VEGETABLES Lian Feng Du1 and·Wen Ke Liu2, 1

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Institute of Plant Nutrition and Resources, Beijing Academy of Agriculture and Forestry Sciences, Beijing, China 2 Environment and Sustainable Development in Agriculture, Chinese Academy of Agricultural Sciences, Key Lab for Agro-Environment and Climate Change, Ministry of Agriculture, Beijing, China

ABSTRACT Two experiments were conducted to investigate the impacts of three antibiotics on seed germination, hydroponic growth and nutritional qualities of vegetables. Seed germination test of three vegetables (cucumber, rape and Chinese cabbage) with six concentrations of tetracycline (TC), chlorotetracycline (CTC) and oxytetracycline (OTC) in form of hydrochlorides was carried out to study the impacts of these antibiotics on root elongation. Hydroponic experiment was designed to explore the growth inhibition and nutritional quality responses of lettuce 

Corresponding author: [email protected].

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Lian Feng Du and·Wen Ke Liu grown in greenhouse with presence of various concentrations of CTC and OTC. The results showed that three antibiotics inhibited root elongation of germinated seeds, and the inhibitory rates depended on the drug species, antibiotic concentrations and vegetable species. Shoot and root biomass of lettuce were significantly reduced when antibiotics added in the nutrient solution. In addition, the soluble sugar content in leaves and petiole, and soluble protein in petiole were modified by antibiotic pollution. To conclude, antibiotic pollution of soil and water will adversely impact the seed germination and growth of protected vegetables grown in soil or hydroponics.

Keywords: Antibiotics, seed germination, vegetable ecotoxicity

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INTRODUCTION Globally, besides as human medicines, an increasing amount of antibiotics as veterinary drugs and feed additives were used annually for disease prevention & treatment and growth promotion in many countries with the rapid development of breeding industry (livestock breeding and aquaculture) (Cromwell et al. 1996; Sarmah et al. 2006; Cabello et al. 2006; Aust et al. 2008). As a result, mostly ingested antibiotics (40-90%) and their metabolites, as emerging persistent contaminants, were excreted with urine and feces, and subsequently disseminated into environmental compartments. Up to date, antibiotic contamination of effluents of sewage treatment plants (Giger et al. 2003; Göbel et al. 2004), surface water (Christian et al. 2003; Kolpin et al. 2002) , manures (Hu et al. 2008; Arikan et al. 2009), soils (Hamscher et al., 2002; Schlüsener et al., 2003; Thiele-Bruhn and Aust 2004) and groundwater (Hamscher et al., 2005) were extensively reported. More importantly, significant amount antibiotics and their bioactive metabolites or degradation products were introduced in agro-ecosystems through antibiotics-polluted animal manures and irrigation water. Nowadays, a large quantity of organic manures were heavily used in protected vegetable production systems (PVPS, e.g. greenhouses and plastic-covered tunnels) and organic vegetable production systems (OVPS) for vegetable production among agro-ecosystems in some countries, e.g. China (Shi et al. 2010; Hu et al. 2010). Therefore, highlevel antibiotics accumulated in soils of PVPS and OVPS as revealed by Hu et al. (2010). As a results, accumulation and transport of antibiotics in soilvegetable systems posssibly poses great risks on crop fitness, soil ecosystems, and quality of plant-based products. Hu et al. (2010) firstly investigated the

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occurrence and seasonality of antibiotics from manure, soil, vegetables and groundwater, and explored the migration of antibiotics from manure to soil, from soil to vegetables and groundwater. Shi et al. (2010) and Hu et al. (2010) firstly put forward the antibiotic pollution issues stemming from organic manures in protected and organic soil-vegetable systems in China. So, ecotoxic impacts of antibiotic contaminants in PVPS and OVPS need to be studied intensively for ensuring the sustainable production. Recently, some results showed that antibiotics impacted seed germination and growth of some crops, and soil enzymatic activities (Jin et al., 2009; Liu et al., 2009). However, there is limited information about the ecotoxic impacts of antibiotics on protected vegetables, even less on hydroponic vegetables. In this study, two experiments were conducted to investigate the impacts of three antibiotics on seed germination, and hydroponic lettuce growth and quality parameters. In the first experiment, seed germination test of three vegetables (cucumber, rape and Chinese cabbage) with six concentrations of tetracycline (TC), chlorotetracycline (CTC) and oxytetracycline (OTC) in form of hydrochlorides was carried out to study the impacts of these antibiotics on seed germination. In the second experiment, hydroponic experiment was designed to explore the growth inhibition and nutritional quality responses of lettuce grown in greenhouse with presence of various concentrations of CTC and OTC.

MATERIALS AND METHODS Materials All the test three veterinary pharmaceuticals, including tetracycline (TC), chlorotetracycline (CTC) and oxytetracycline (OTC) in form of hydrochloride, were purchased from Sigma-Aldrich Chemical Co. Four vegetable species were used in two experiments, including cucumber, Chinese cabbage, lettuce and rape, and the cultivars used are Jingyu1, Jinlü80, Italian cultivar and Beiji, respectively.

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Experiment 1 Impacts of three antibiotics (tetracycline hydrochloride, chlorotetracycline hydrochloride and oxytetracycline hydrochloride) on germination of cucumber (Jingyu1), Chinese cabbage (Jinlü80) and rape (Beiji) were investigated in experiment 1. In the experiment, six concentrations of antibiotics were designed, including 0, 10, 50, 100, 250, 500 mg/L, respectively. Fifty-four treat-ments were included, and each treatment replicated three times. Eight seeds for cucumber and ten seeds for Chinese cabbage and rape were disinfected by 10% H2O2 for ten minutes. After rinsing fully by tap water, seven seeds for cucumber and ten seeds for Chinese cabbage and rape were placed on a filter paper which was moistened with drug solutions and set down in a Petri dish (diameter 9cm). All the Petri dishes were cultured in a dark culture chamber. The temperature of the dark culture chamber were controlled within 30±0.5℃. The fourth day after cultivation, the root length for each replicate was recorded and averaged, then root length of three replicates of each drug concentration treatment was averaged again. Using the averages of all treatments, inhibitory rates were calculated as followed: The inhibitory rate (IR) of the three vegetables was calculated as followed: Inhibitory rate (IR) = (Root elongation in control treatment-Root elongation in drug treatment)/ Root elongation in control treatment.

Experiment 2 Impacts of CTC and OTC on growth and quality parameters of hydroponic lettuce were investigated in experiment 2. Five treatments were designed, including 0, 2.5 and 5.0 mg/L CTC (CK, CTC-2.5 and CTC-5), and 2.5, 5.0 mg/L OTC (CTC-2.5 and CTC-5), respectively. All antibiotics were fully dissolved in water and then mixed into nutrition solution. Each treatment replicated three times. The hydroponic nutrient solutions were prepared with distilled water and analytical reagents. The nutrient solution is composed of 0.75 K2SO4, 0.5 KH2PO4, 0.65 MgSO4, 0.1 KCl, 5.0 Ca（NO3）2, 1.0×10-3 H3BO3, 1.0×10-3 MnSO4,1.0×10-4 CuSO4, 5.0×10-6 (NH4)6MO7O24, 1.0×103 ZnSO4, 0.1 EDTA-Fe (mmol/L). The experiment was carried out in a glasshouse, with a temperature ranging from 15 to 30℃.

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Figure 1. Inhibitory rates of tetracycline on root elongation of three vegetable seeds.

Lettuce（Lactuca sativa）, an Italian cultivar, was transplanted and cultivated in the experiment pot (32cm×24cm×11cm) supplied with 6L nutrient solution after 20 days cultivation in a seedling tray. Two lines and six holes were made on the lid at an equably distance, and a central one was used as a gasvent to aerate the nutrient solution by a air-pump (Atman EP-9000）to ensure the supply of soluble oxygen around the roots. Five lettuces in one hydroponic pot were planted. The experiment started on Sept. 13, 2010. The lettuces were harvest after thirty days. Lettuce shoot was separated three parts, i.e. new leaf, expanded leaf and petiole for nitrate concentration determination. Also the SPAD of new and expanded leaf were determined with Minolta SPAD-502 chlorophyll meter (made in Japan). The soluble sugar content and soluble protein content in leaf and petiole were measured by the methods described in Li et al. (2000). Also the SPAD of new and expanded leaf were determined with Minolta SPAD-502 chlorophyll meter (made in Japan).

RESULTS Impacts of Antibiotics on Root Elongation of Three Vegetables Impacts of TC on Root Elongation of Three Vegetables The TC inhibited the root elongation of cucumber, rape and Chinese cabbage, and the inhibitory rates grew with the increase of the drug concen-

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tration (Figure 1). The tolerance of Chinese cabbage seed to three antibiotics is higher than cucumber and rape, particularly at the low drug concentration (